Carrier based drug delivery system

ABSTRACT

The present invention relates to a novel drug delivery system of use in the improved delivery of drug therapeutic agents into target cells. The system comprises a drug moiety linked to a carrier moiety wherein the carrier moiety comprises a homeobox peptide or a fragment or derivative thereof.

RELATED APPLICATIONS

This application is a continuation application of Ser. No. 09/346,847filed on Jul. 2, 1999, and issued as U.S. Pat. No. 6,472,507B1 on Oct.29, 2002, which claims benefit of GB 98/14527.9 filed on Jul. 3, 1998.The contents of all of the aforementioned application(s) are herebyincorporated by reference.

BACKGROUND

The pharmaceutical industry has for many years concerned itself with theefficient delivery of therapeutic agents. This problem may be attributedto the short clearance time of the agent in the body (short half-life),the location of the site of action or possibly the nature of thetherapeutic agent itself, for example, its solubility, hydrophobicityetc. Thus, many developments and strategies have been adopted, includingformulating the therapeutic agent so as to protect it from a hostileenvironment on route to its site of action, by for example, entericallycoated tablets, controlled release devices and the like.

The development of peptide derived therapeutic agents has posed afurther problem due their susceptibility to enzymatic degradation notonly in the GI tract but also in the bloodstream. An example of how thisproblem has been addressed relates to the incorporation of the peptidesinto liposomes or polymeric microspheres that target the peptides to thelymph system.

A further related problem, especially for therapeutic agents thatfunction intracellularly is the barrier posed by the cell membrane.Thus, it may be possible to increase the half life of the agent orensure that it passes through the body without being degraded, but manyagents must actually enter cells to exert their therapeutic effect.

European Patent 485578 discloses that the homeodomain and specifically,helix 3 of a homeobox peptide, particularly that derived from theDrosophila Antennapedia, is of use as an intracellular transport vector.The patent disclosed that a specific 57 amino acid sequence of aDrosophila Antennapedia homeopeptide (referred to as the pAntp peptide)was capable of penetrating fibroblasts and embryo cells (in vivo).Emphasis was placed upon the last 27 amino acids of the sequence thatcorrespond with the helix 3 and 4. There is no description of the pAntppeptide being linked to any other peptide or therapeutic agent.

Subsequent disclosures (Derossi D et al., J Biol Chem (1994) 269,10444-10450, Joliot A H et al., (1991) The New Biol 3, 1121-1134 andPNAS (1991) 88, 1864-1868, Perez F et al., J Cell Sci (1992) 102,712-722), all disclose how a 16 amino acid synthetic peptide derivedfrom the third helix of the Antennapedia homeodomain may be used for theintracellular delivery of bioactive products and antisenseoligonucleotides. The amino acid sequence of use is RQIKIFQNRRMKWKK (SEQID No. 1) also known as Penetratin®.

In an effort to prevent the enzymatic cleavage of this peptide BrugidouJ et al., (Biochem Biophys Res Comm (1995) 214(2), 685-693) prepared aretro-inverso form (D amino acids in reverse order) of SEQ ID No. 1,substituting the two isoleucine resides at positions 3 and 5 ofpenetratin with valine and adding a glycine residue at the C-terminus tofacilitate binding to a resin. A further retro-inverso form was preparedreplacing the extra glycine with a cholesterol moiety attached via asulfhydryl linker group. The addition of the cholesterol moiety improvedpenetration due to the increased hydrophobicity of the molecule.

This development of the retro-inverso form of penetratin has given riseto WO 97/12912 that discloses peptides of 16 amino acids comprisingbetween 6 and 10 hydrophobic amino acids wherein the sixth amino acidfrom either end is tryptophan. This disclosure attempts to define theminimal characteristics of sequences capable of acting asinternalisation vectors.

Penetratin, its analogues and retro-inverso forms have therefore beendescribed as being of use as a carrier to facilitate the cellularinternalisation of conjugated peptides or oligonucleotides.

SUMMARY OF THE INVENTION

The present invention aims to provide a delivery system for therapeuticdrugs that is capable of facilitating the internalisation of the druginto cells, thus enhancing the delivery and/or therapeutic effect of thedrug. The delivery system may also improve the half-life of the drug inthe human or animal body, improve its solubility in biological fluids,minimise known toxic or non-desirable side-effects, enhance the onset ofaction of the desired therapeutic effect, provide alternative routes forthe administration of the drug, enhance the biodistribution andmetabolism of the drug moiety and decrease the incidence of drugresistance.

The present invention relates to a novel drug delivery system of use inthe improved delivery of drug therapeutic agents into target cells. Thedelivery system provides other benefits that include enhancement interms of the metabolism, distribution and excretion of the drug. Thedelivery system may be therapeutically active in both its intact anddissociated states.

Thus, the invention relates to a delivery system comprising a drugmoiety linked to a carrier moiety comprising a homeobox peptide or afragment or derivative thereof. As is discussed hereinafter, the drugmoiety is not a peptide or oligonucleotide and the carrier moiety may bea derivative of penetratin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the comparison of cell internalisation of a delivery systemof the present invention compared to the carrier moiety alone.

FIG. 2 shows the intracellular stability of a delivery system of thepresent invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a first embodiment, the delivery system comprises a drug moietylinked to a carrier moiety. The drug moiety may be directly orindirectly linked to the carrier moiety. In the preferred embodimentwherein the drug moiety is indirectly linked to the carrier, the linkagemay be by an intermediary bonding group such as a sulphydryl or carboxylgroup or any larger group, all such linking groups and others describedbelow, are hereinafter referred to as linker moieties.

In accordance with the present invention, suitable drug moieties includeany therapeutically active non-peptide/oligonucleotide drug. Thus, thedrug moiety may be selected from cytotoxic agents, anti-neoplasticagents, anti-hypertensives, cardioprotective agents, anti-arrhythmics,ACE inhibitors, anti-inflammatory's, diuretics, muscle relaxants, localanaesthetics, hormones, cholestrol lowering drugs, anti-coagulants,anti-depressants, tranquilizers, neuroleptics, analgesics such as anarcotic or anti-pyretic analgesics, anti-virals, anti-bacterials,anti-fungals, bacteriostats, CNS active agents, anti-convulsants,anxiolytics, antacids, narcotics, antibiotics, respiratory agents,anti-histamines, immunosuppressants, immunoactivating agents,nutritional additives, anti-tussives, diagnostic agents, emetics andanti-emetics.

Preferably the drug moiety is a cytotoxic or anti-neoplastic agent,particularly those which are used for cancer therapy or such drug inphotoactivatable form. Such drugs include, in general, DNA damagingagents, anti-metabolites, anti-tumour antibiotics, natural products andtheir analogues, dihydrofolate reductase inhibitors, pyrimidineanalogues, purine analogues, cyclin-dependent kinase inhibitors,thymidylate synthase inhibitors, DNA intercalators, DNA cleavers,topoisomerase inhibitors, anthracyclines, vinca drugs, mitomycins,bleomycins, cytotoxic nucleosides, pteridine drugs, diynenes,podophyllotoxins, platinum containing drugs, differentiation inducers,and taxanes. Particularly useful members of those classes include, forexample, methotrexate, methopterin, dichloromethotrexate,5-fluorouracil, 6-mercaptopurine, tri-substituted purines such asolomoucine, roscovitine, bohemine and purvalanol, flavopiridol,staurosporin, cytosine arabinoside, melphalan, leurosine, actinomycin,daunorubicin, doxorubicin, mitomycin D, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin (and derivatives thereof),etoposide, cisplatin, carboplatinum, vinblastine, vincristine, vindesin,paclitaxel, docetaxel, taxotere retinoic acid, butyric acid, acetylspermidine, tamoxifen, irinotecan and camptothecin. Most preferably thedrug moiety is selected from methotrexate, podophyllotoxin (andderivatives thereof), etoposide, camptothecin, paclitaxel, doxorubicin,roscovitine and bohemine.

The carrier moiety as used in the present invention may be any moietythat is capable of facilitating the cellular internalisation of the drugmoiety. Suitable carrier moieties include homeobox peptides orderivatives thereof such as the helix 3 of a homeobox peptide.Preferably, the homeobox peptide is derived from the DrosophilaAntennapedia homeoprotein, sequences homologous thereto or derivativesthereof. More preferably, the carrier moiety is penetratin or aderivative thereof. Derivatives of penetratin have been described in theliterature, for example EP 485578B, that discloses sequences homologousto pAntp. Further derivatives of penetratin that may be utilised in thepresent invention include truncated forms and/or modified forms ofpenetratin described in WO97/12912, UK Patent Applications 9825000.4filed 13 Nov. 1998 and 9902522.3 filed 4 Feb. 1999 the contents of whichare hereby incorporated by reference. A preferred truncated form ofpenetratin is RRMKWKK (SEQ ID No. 2). Further truncated forms includemoieties of up to 15 amino acid residues including the sequences such asNRRMKWKK (SEQ ID NO. 3), QNRRMKWKK (SEQ ID No. 4) and FQNRRMKWKK (SEQ IDNo. 5) or more preferably a 7 amino acid peptide selected from KRMKWKK(SEQ ID No. 6), RKMKWKK (SEQ ID No. 7), RREKWKK (SEQ ID No. 8), RRQKWKK(SEQ ID No. 9), RROKWKK (SEQ ID No. 10), RRMKQKK (SEQ ID No. 11),RRMKWFK (SEQ ID No. 12), RORKWKK (SEQ ID No. 13), RRMWKKK (SEQ ID No.14) and RRMKKWK (SEQ ID No. 15) (using standard single amino acidnotation, ornithine (O), diaminobutyric acid (B), norleucine (N)).

Within the carrier moieties defined as penetratin or derivativesthereof, a further modification that is beneficial in the context of thepresent invention is conversion of the free carboxyl group of thecarboxy terminal amino acid residue, to an carboxamide group. By way ofexample, when the carrier moiety is penetratin (SEQ ID No. 1) thecarboxy terminal lysine residue may have its carboxyl group convertedinto an carboxamide group. This modification is believed to enhance thestability of the carrier moiety and hence the delivery system as awhole.

The carrier moiety may be in the L or D optically active form. As usedherein, when no indication is given, the carrier is in the L form.D-penetratin is described in Brugidou J et al., (Biochem Biophys ResComm (1995) 214(2), 685-693). The carrier moiety may also be arranged inthe retro form, i.e with the amino acid residues in the reverse order totheir parent sequence. Such retro forms may also exist in L and D forms.Thus, in a further preferred embodiment the carrier moiety may beD-penetratin or the D form of the truncated and/or modified formsdiscussed above.

The drug moiety may be attached to either end of the carrier moiety e.g.if the carrier moiety is penetratin as shown in SEQ ID No. 1 or aderivative thereof, the drug moiety may be directly or indirectlyattached to the terminal lysine or arginine residues. Preferably, thedrug moiety is attached to the amino terminal end of the carrier.

As discussed above the drug and carrier moieties may be linked directlyor indirectly via a linker moiety. Direct linkage may occur through anyconvenient functional group on the drug moiety such as a hydroxy,carboxy or amino group. Indirect linkage which is preferable, will occurthrough a linking moiety. Suitable linking moieties include bi- andmulti-functional alkyl, aryl, aralkyl or peptidic moieties, alkyl, arylor aralkyl aldehydes acids esters and anyhdrides, sulphydryl or carboxylgroups, such as maleimido benzoic acid derivatives, maleimido proprionicacid derivatives and succinimido derivatives or may be derived fromcyanuric bromide or chloride, carbonyldiimidazole, succinimidyl estersor sulphonic halides and the like. The functional groups on the linkermoiety used to form covalent bonds between linker and drugs on the onehand, as well as linker and carrier moiety on the other hand, may be twoor more of, e.g., amino, hydrazino, hydroxyl, thiol, maleimido,carbonyl, and carboxyl groups, etc. The linker moiety may include ashort sequence of from 1 to 4 amino acid residues that optionallyincludes a cysteine residue through which the linker moiety bonds to thecarrier moiety.

Preferably, the linker moiety includes a cysteine residue that providesthe actual linkage to the carrier moiety such as to form a linkage ofthe type drug-(linker-Cys)-carrier. Within the context of thespecification this cysteine residue is considered as a component of thelinker moiety. Thus, the complete linker moiety may only be formed as aresult of the drug-carrier coupling reaction as the cysteine residuecomponent of the linker may be conveniently prepared as part of thecarrier moiety. In a preferred embodiment the linker moiety is selectedfrom (methylamino)benzoyl-Cys, succinimidobenzoyl-Cys,succinimidopropionoyl-Cys, β-alanyl-succinyl, acetyl-Cys and(4″-aminoanilino)-succinimidopropionoyl-Cys. In such preferredembodiments, the cysteine residue preferably originates as a terminalresidue of the carrier moiety, whereas the non-cysteine component of thelinker is coupled to the drug moiety prior to reaction with the carrier.The complete linker moiety is therefore only formed upon reacting thedrug and carrier moieties together.

In a manner identical to the inclusion of a cysteine residue into thelinker moiety, further amino acid residues may be included in the linkerwhich like the cysteine residue form the connection with the carriermoiety. For example, 3 or 4 amino acid residues may be included andthese preferably include the cysteine residue discussed above. Any aminoacid residues may be included, it is however preferable to select theresidues from cysteine, β-alanine and glycine. The inclusion of suchresidues is preferable, particularly including cysteine, when thecarrier moiety is a truncated form of penetratin such as RRMKWKK (SEQ IDNo. 2).

In use, the delivery system may dissociate by way of chemical orenzymatic cleavage between the drug and carrier moieties. Within theembodiments wherein the linker moiety includes amino acid residues, suchcleavage may occur within the linker moiety itself.

In accordance with the present invention each carrier moiety is linkedto at least one drug moiety. In a further embodiment, the carrier moietyis prepared such as to facilitate linkage to more than one drug moiety,each drug moiety being the same or different. For example, the carriermoiety may comprise components that themselves facilitate the attachmentof more than one drug moiety such as derivatives of naturally occurringamino acids or insertion of a multi-valent synthetic amino acid, or itmay be specifically adapted to do so for example by a network ofbranched lysine residues that may be attached to the carrier moiety as alinking group and each lysine residue may then be attached to a drugmoiety. In this manner a single carrier moiety may carry up to 32 drugmoieties, preferably from 2 to 10 or more preferably from 4 to 5 drugmoieties. In this further embodiment each drug moiety may be directly orindirectly linked to the carrier moiety by the same or different linkermoiety. When more than one different type of drug moiety is attached, itis possible to co-ordinate the ratios and dosages of the individualdrugs to facilitate the administration of specific drug combinations.

Preferred examples of this embodiment include when the carrier moiety ispenetratin with a network of lysine residues attached to at least oneend facilitating the attachment of up to 32 drug moieties or when thecarrier moiety is penetratin or a derivative thereof, such as SEQ ID No.2 (truncated 7-mer), the linker moieties are succinimidopropionyl andthe drug moieties are selected from podophyllotoxin (at both ends of thecarrier moiety) or epipodophyllotoxin together with either camptothecinor paclitaxel.

In a particularly preferred embodiment of the invention, the carriermoiety is penetratin or a derivative thereof that is indirectly linkedto a drug moiety selected from doxorubicin, methotrexate,podophyllotoxin (and derivatives thereof), etoposide, camptothecin,paclitaxel, doxorubicin, roscovitine and bohemine.

In a further embodiment of the invention, the delivery system mayfurther comprise a targeting moiety. The targeting moiety is capable ofdirecting the delivery system to the specific cell type to which it ispreferable for the drug moiety to function. Thus, the targeting moietyacts as an address system biasing the bodies natural distribution ofdrugs or the delivery system to a particular cell type. The targetingmoiety may be attached to the drug moiety or more preferably to thecarrier moiety.

Suitable targeting moieties include the peptide sequences identified byE Ruoslahti et al. in U.S. Pat. No. 5,622,699; Pasqualini, R, Ruoslahti,E. Nature (London) (1996), 380, 364-366, Ruoslahti, E. Ann. Rev. CellDev. Biol. (1996), 12, 697-715; Arap, W, Pasqualini, R, Ruoslahti, E,Science (1998), 279, 377-380. These disclosures, which are hereinincorporated by reference, described certain peptides that have beenfound to act as address labels to certain cell types. Such peptides whenattached to either the drug or more preferably, the carrier moiety willdirect the delivery system, upon arrival at which the carrier moietywill facilitate the cellular internalisation of the drug moiety.

The delivery systems described herein are novel chemical entities.Specific chemical entities disclosed herein include;

# Drug moiety Linker moiety Carrier moiety (methotrexate)₄((methylamino)- (L)₃βARQIKIW- benzoyl-EGβA)₄ FQNRRMKWKK- OH (SEQ ID No.16) doxorubicin succinimido- RQIKIWFQNRR- benzoyl-C MKWKK-OH doxorubicinsuccinimido- (D-K)(D-K)(D-W)- benzoyl-C (D-K)(D-M)(D-R)-(D-R)(D-N)(D-Q)- (D-F)(D-W)(D-I)- (D-K)(D-I)(D-Q)- (D-R-NH₂) paclitaxel2′-succinimido- RQIKIWFQNRRM- propionoyl-C KWKK-OH N-term paclitaxel2′-succinimido- RQIKIWFQNRRM- C-term carboxy- propionoyl-GCG KWKKfluorescein βA paclitaxel 2′-succinimido- RQIKIWFQNRRM- propionoyl-CKWKK-NH₂ paclitaxel 2′-succinimido- RRMKWKK-NH₂ propionoyl-CβApaclitaxel 7-succinimido- RQIKIWFQNRRM- propionoyl-C KWKK-OHpodophyllotoxin 4-succinimido- RQIKIWFQNRRM- propionoyl-C KWKK-OH N-termpodophyllotoxin 4-succinimido- RQIKIWFQNRRM- C-term biotinamidocaproylpropionoyl-GCG KWKK βA podophyllotoxin 4-succinimido- RQIKIWFQNRRM-propionoyl-C KWKK-NH₂ podophyllotoxin 4-succinimido- (D-R)(D-Q)(D-I)-propionoyl-C (D-K)(D-I)(D-W)- (D-F)(D-Q)(D-N)- (D-R)(D-R)(D-M)-(D-K)(D-W)(D-K)- (D-K-NH₂) podophyllotoxin 4-succinimido- RRMKWKK-NH₂propionoyl-CβA podophyllotoxin 4-succinimido- (D-R)(D-R)(D-M)-propionoyl-CβA (D-K)(D-W)(D-K)- (D-K-NH₂) epipodophyllotoxin4′-succinimido- RQIKIWFQNRRM- propionoyl-C KWKK-OH epipodophyllotoxin4′-succinimido- RQIKIWFQNRRM- propionoyl-C KWKK-NH₂ epipodophyllotoxin4′-succinimido- RRMKWKK-NH₂ propionoyl-CβA 4′-demethyl 4-succinimido-RQIKIWFQNRRM- epipodophyllotoxin propionoyl-C KWKK-OH etoposide (G2, G3succinimido- RQIKIWFQNRRM- and 4′) propionoyl-C KWKK-OH roscovotinesuccinimido- RQIKIWFQNRRM- propionoyl-C KWKK-OH bohemine βA-succinyl-βARQIKIWFQNRRM- KWKK-OH bohemine succinimido- RQIKIWFQNRRM- propionoyl-CKWKK-OH podophyllotoxin 4-acetyl-C RQIKIWFQNRRM- KWKK-OH podophyllotoxin4-acetyl-CβA RRMKWKK-NH₂ 4′-demethyl 4-acetyl-CβA RRMKWKK-NH₂epipodophyllotoxin 4′-demethyl 4-acetyl-C RQIKIWFQNRRM-epipodophyllotoxin KWKK-NH₂ podophyllotoxin 4-succinimido- RRMKWKK-NH₂propionoyl-GCβA camptothecin 10-O-succinimido- RQIKIWFQNRRM-propionoyl-C KWKK-NH₂ C-term podophyllotoxin 4-succinimido- RRMKWKKpropionoyl-C N-term podophyllotoxin 4-succinimido- propionoyl-C N-termepipodophyllotoxin 4′-succinimido- RRMKWKK propionoyl-C C-termcamptothecin 10-O-succinimido- propionoyl-C N-term epipodophyllotoxin4′-succinimido- RRMKWKK propionoyl-C C-term paclitaxel 2′-(succinimido)-propionoyl-C 4′-methoxy- 4-(4″-aminoanilino) RQIKIWFQNRRM-epipodophyllotoxin succinimido- KWKK-NH₂ propionoyl-C 4′-methoxy-4-(4″-aminoanilino) RRMKWKK-NH₂ epipodophyllotoxin succinimido-propionoyl-CβA 4′-demethyl- 4-(4″-aminoanilino) RRMKWKK-NH₂epipodophyllotoxin succinimido- propionoyl-CβA (RQIKIWFQNRRMKWKKcorresponds with SEQ ID No. 25 and RRMKWKK corresponds with SEQ ID No.26)The therapeutic effect resulting from the administration of the deliverysystem may arise from the intact delivery system or any of itsdissociated components that include the drug moiety i.e the drug moietyalone or bound to the linker, part of the linker or the linker and partof the carrier. Thus the term “delivery system” has been used herein tohave its ordinary meaning i.e that of delivering something such as thedrug moiety and additionally to include the system or any portionthereof as being active in its intact state. Thus, the benefits providedby the system discussed above are applicable to the drug and deliverysystem.

The delivery systems described herein can be macromolecules. Suchmacromolecules can be selected from any of the delivery systems definedherein.

The delivery vectors may be prepared by any methods known in the art.For example, the pAntp peptide can be assembled using conventionalsolution- or solid-phase peptide synthesis methods, affording a fullyprotected precursor with only the terminal amino group in deprotectedreactive form. This function can then be reacted directly with a drugmoiety or a suitable reactive derivative of a drug moiety.Alternatively, this amino group may be converted into a differentfunctional group suitable for reaction with a drug moiety or a linker.Thus, e.g. reaction of the amino group with succinic anhydride willprovide a selectively addressable carboxyl group, while further peptidechain extension with a cysteine derivative will result in a selectivelyaddressable thiol group. Once a suitable selectively addressablefunctional group has been obtained in the delivery vector precursor, adrug moiety or a derivative thereof may be attached through e.g. amide,ester, or disulphide bond formation. Alternatively, a linker group, e.g.m-maleimidobenzoyl, is introduced by reaction of a linker groupprecursor with the selectively addressable function of the deliveryvector precursor, followed by formation of a covalent bond between thelinker group and a drug moiety. Multivalent drug-delivery vectorconstructs may be obtained, inter alia, by successive extension of theselectively addressable delivery vector precursor with trivalentchemical groups. Thus peptide chain extension with e.g.N^(α,ε)-Fmoc-protected Lys derivatives will afford di-, tetra-, andocta-valent construct precursors after one, two, or threecoupling/Fmoc-deprotection cycles.

Using these methods, the skilled person will be capable of preparing awide variety of drug-carrier conjugates utilising a variety of linkermoieties. As exemplified below, an appropriate group on the drug moietymay be selected for attachment to the carrier moiety and if desired alinker joined to the drug or carrier moiety, or both prior to theircoupling.

The compounds of the present invention may be formulated with aphysiologically acceptable diluent or carrier for use as pharmaceuticalsfor both veterinary, for example in mammals, and particularly human useby a variety of methods. For instance, they may be applied as acomposition incorporating a liquid diluent or carrier, for example anaqueous or oily solution, suspension or emulsion, which may often beemployed in injectable form for parental administration and thereforemay conveniently be sterile and pyrogen free. Oral administration mayalso be used and although compositions for this purpose may incorporatea liquid diluent or carrier, it is more usual to use a solid, forexample a conventional solid carrier material such as starch, lactose,dextrin or magnesium stearate. Such solid compositions may take the formof powders but are more conveniently of a formed type, for example astablets, cachets, or capsules (including spansules). Alternative, morespecialized types of formulation include liposomes and nanoparticles.

Other types of administration than by injection or through the oralroute which are of use in both human and veterinary contexts include theuse of suppositores or pessaries. Another form of pharmaceuticalcomposition is one for buccal or nasal administration or administrationto the airways such as alveolar tissue. Other formulations of topicaladministration include lotions, ointments, creams, gels and sprays.

Compositions may be formulated in unit dosage form, i.e. in the form ofdiscrete portions containing a unit does, or a multiple or sub-unit of aunit dose.

As is described in the Examples below, the delivery system of thepresent invention provides several advantages over known deliverysystems for non-peptide/oligonucleotide delivery systems. Theseadvantages include improved efficacy compared to conventionaltreatments, improved cellular uptake of the therapeutic agent, improvedwater solubility, reduction of side effects and cellular bioavailabilityand decreased occurrence of drug resistance.

The contents of all references, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES

Abbreviations

Amino acid and peptide nomenclature conforms to IUPAC-IUB rules (Eur. J.Biochem. 1984, 138, 9-37). Other abbreviations: AcOH, acetic acid; Boc,tert.-butyloxycarbonyl; Bu^(t), tert.-butyl; DE MALDI-TOF MS, delayedextraction matrix-assisted laser desorption ionisation time-of-flightmass spectrometry, DIC, 1,3-diisopropylcarbodiimide; DIEA,diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMEM, Dulbecco'smodified Eagle medium; DMF, dimethylformamide; Et₃N, triethylamine;EtOAc, ethyl acetate; Et₂O, diethyl ether, FCS, foetal calf serum; HOBt,1-hydroxybenzotriazole; MeCN, acetonitrile; MeOH, methanol; NMR, nuclearmagnetic resonance spectroscopy; PE, petroleum ether 40-60° C. boilingfraction; PBS, phosphate-buffered saline; Pmc,2,2,5,7,8-pentamethylchroman-6-sulfonyl; PyBOP,benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate;RP-HPLC, reversed-phase high-performance liquid chromatography; TFA,trifluoroacetic acid; Trt, triphenylmethyl.

General

RP-HPLC was conducted using Vydac 218TP54 (4.5×250 mm) and 218TP 1022(22×250 mm) columns for analytical and preparative purposes,respectively. Flow rates were 1 mL/min for analytical and 9 mL/min forpreparative runs. Gradient elution (constant 25° C.) was performed usingincreasing amounts of MeCN in water (containing a constant concentrationof 0.1% TFA) over 20 min (analytical) or 40 min (preparative). Flashchromatography was carried out as described (W. C. Still, M. Kahn, A.Mitra, J. Org. Chem., 1978, 43, 2923-2925) using Merck silica gel 60,230-240 mesh. Peptide synthesis was carried out using an ABI 433APeptide Synthesizer (Perkin-Elmer Applied Biosystems). Amino acidderivatives were from Novabiochem AG, Läufelfingen, Switzerland, exceptFmoc-D-Ile-OH, which was from Bachem AG, Bubendorf, Switzerland.Standard synthesis protocols (0.1 mmol or 0.25 mmol scale “FastMocMonPrevPk” programs) based on the Fmoc-protection strategy (G. B.Fields, R. L. Noble, Intl. J. Peptide Protein Res., 1990, 35, 161) wereapplied. Peptidyl resins were cleaved and deprotected using thefollowing reagent: 0.75:0.5:0.5:0.25:10 (w/v/v/v/v) phenol, water,thioanisole, 1,2-ethanedithiol, TFA (D. S. King, C. G. Fields, G. B.Fields, Intl. J. Peptide Protein Res., 1990, 36, 255). DE MALDI-TOF MSwas performed using a Dynamo (Thermo BioAnalysis, Hemel Hempstead,England) spectrometer. The matrix used was a-cyano-4-hydroxycinnamicacid. The spectrometer was calibrated using authentic peptides withappropriate masses. NMR spectra were recorded on a Brucker DPX300instrument. Paclitaxel, podophyllotoxin, and 10-hydroxycamptothecin werefrom Hande Tech Development Co. USA Inc, Houston, Tex., USA.4′-Demethylepipodophyllotoxin was prepared as described (M. Kuhn, C.Keller-Juslén, A. von Wartburg, Helv. Chim. Acta, 1969, 52, 944).Roscovitine was prepared essentially as described (L. Havlicek, J.Hanus, J. Vesely, S. Leclerc, L. Meijer, G. Shaw, M. Strnad, J. Med.Chem. 1997, 40, 408). Bohemine(6-(benzylamino)-2-[(3-(hydroxy-propyl)amino]-9-isopropylpurine) wassynthesised similarly. Anhydrous DMF, ClCH₂CH₂Cl, and CH₂Cl₂, storedover molecular sieve 4A, were used throughout.

Example 1H-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-resin(SEQ ID No. 25)

The peptide sequence was assembled onFmoc-Lys(Boc)-[(4-(hydroxymethyl)pheneoxyacetyl)-resin] (ABI 401425; 0.5mmol/g). The final peptidyl resin (1.37 g, 100%) was washed with Et₂Oand dried in vacuo. In order to demonstrate the chemical integrity ofthis intermediate, a small aliquot of peptidyl resin was cleaved anddeprotected, followed by analysis of the crude productH-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 25), which revealed purity of >90% (anal. RP-HPLC) andchemical identity (DE MALDI-TOF MS and quantitative amino acidanalysis).

[H-Glu(OBu^(t))-Gly-bAla]₄-Lys₂-Lys-bAla-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Glu(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-resin(SEQ ID No. 28)

The above peptidyl resin (137 mg, 25 μmol) was acylated withFmoc-βAla-OH (47 mg, 0.15 mmol), PyBOP (78 mg, 0.15 mmol), HOBt (20 mg,0.15 mmol) and DIEA (39 μL, 0.225 mmol) in DMF (2 mL) during 2 h. It wasthen Fmoc-deprotected with 20% piperidine in DMF for 20 min and washedextensively with DMF. The product was further extended by two successiveacylation and deprotection cycles using Fmoc-Lys(Fmoc)-OH (0.15 mmol infirst cycle; 0.3 mmol in second cycle) using similar coupling anddeprotection steps. This was followed by further chain extension withFmoc-Gly-OH (0.6 mmol) and Fmoc-Glu(OBu^(t))-OH (0.6 mmol), again usingsimilar acylation and Fmoc-deprotection conditions. The product wasFmoc-deprotected and washed extensively with DMF, CH₂Cl₂ and Et₂O,followed by drying in vacuo. In order to demonstrate chemical integrityof this intermediate, a small aliquot of peptidyl resin was cleaved andside-chain deprotected, followed by analysis of the crude product[H-Glu-Gly-βAla]₄-Lys₂-Lys-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 28), which revealed purity (>89%; RP-HPLC, 15-25% MeCNgradient, t_(R)=17.7 min, λ=200-300 nm) and identity (DE MALDI-TOF MS:[M+H]⁺=3732, C₁₆₅H₂₆₉N₅₃O₄₄S=3731.30).

{[4[N-(2,4-diamino-6-pteridinyl-methyl)-N-methylamino]benzoyl]-Glu(OBu^(t))-Gly-βAla}₄-Lys₂-Lys-βAla-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-resin(SEQ ID No. 28)

The above peptidyl resin (76 mg, 25 μmol) was reacted overnight at roomtemperature with4[N-(2,4-diamino-6-pteridinyl-methyl)-N-methylamino]benzoic acidhemihydrochloride dihydrate (76 mg, 0.2 mmol) and PyBOP (104 mg, 0.2mmol), HOBt (27 mg, 0.2 mmol) and DIEA (70 μL, 0.4 mmol) in DMF (2 mL).The product was washed successively with DMF, CH₂Cl₂ and Et₂O and driedin vacuo to afford the title compound (85 mg orange peptidyl resin).

{[4[N-(2,4-Diamino-6-pteridinyl-methyl)-N-methylamino]benzoyl]-Glu-Gly-βAla}₄-Lys₂-Lys-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 28)

The above product was cleaved and deprotected (12 mL cleavage reagent,1.5 h). Resin residue was then filtered off and washed on a sinter withsmall aliquots of neat TPA. The combined filtrate and washings weretreated with Et₂O (100 mL) and cooled. The precipitated product wascollected by centrifugation and the ethereal supernatant was decanted.The product was washed three more times with Et₂O in a similar fashion.The final crude product was dried in vacuo (61 mg orange powder). Thismaterial was redissolved in 0.1% aq TFA (4 mL) and filtered. Theresulting solution was applied (two separate runs) to a preparativeRP-HPLC column (17.5-27.5% MeCN gradient runs). Peak fractions werecollected, monitored (analytical RP-HPLC) and pooled as appropriate.After vacuum centrifugation, pure title compound (13.5 mg) was obtained.Anal. RP-HPLC: t_(R)=17.8 (17.5-27.5% MeCN gradient; purity>99%,f=200-300 nm). DE MALDI-TOF MS: [M+H]⁺=4962 (C₂₂₅H₃₂₁N₈₁O₄₈S=4960.54).

Example 2H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID) No. 17)

H-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-resin(SEQ ID No. 1) (see example 1; 411 mg, 75 μmol) was acylated withFmoc-Cys(Trt)-OH (264 mg, 0.45 mmol), PyBOP (234 mg, 0.45 mmol), HOBt(61 mg, 0.45 mmol) and DIEA (0.12 ml., 0.675 mmol) in DMF (3 mL) during3 h. The resulting peptidyl resin was washed with DMF (3×5 min, 25 mLeach), drained and treated with 20% piperidine in DMF during 20 min.After filtration of the reagent, the productH-Cys(Trt)-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-resin(SEQ ID No. 27) was washed successively with DMF, CH₂Cl₂ and Et₂O,before being dried in vacuo. This product was cleaved/deprotected (2 h).Resin residue was then filtered off and washed on a sinter with smallaliquots of neat TFA. The combined filtrate and washings were treatedwith Et₂O (100 mL) and cooled. The precipitated product was collected bycentrifugation and the ethereal supernatant was decanted. The productwas washed three more times with Et₂O in a similar fashion. The finalcrude product was dried in vacuo (238 mg). An aliquot (119 mg) of thismaterial was redissolved in 0.1% aq TFA (2 mL) and filtered. Theresulting solution was purified by preparative RP-HPLC (17.5-27.5% MeCNgradient). Peak fractions were collected, monitored (analytical RP-HPLC)and pooled as appropriate. After vacuum centrifugation, pure titlecompound (60.9 mg) was obtained. Anal. RP-HPLC: t_(R)=15.8 min(17.5-27.5% MeCN gradient; purity>99%, λ=214 nm). DE MALDI-TOF MS:[M+H]⁺=2351 (C₁₀₇H₁₇₃N₃₅O₂₁S₂=2349.87).

N-[3-(Maleimido)benzoyl]doxorubicin

Doxorubicin hydrochloride (5.9 mg, 10 μmol) was dissolved in water (1mL) and DMF (0.5 mL). Buffer (0.1 M aq phosphate, pH 7.2; 0.5 mL) wasadded with stirring. To the resulting suspension 3-maleimidobenzoic acidN-hydroxysuccinimide ester (12.9 mg, 40 μmol) in DMF (1 mL) was addeddropwise. The red-coloured reaction mixture cleared temporarily andafter ca. 10 min precipitation was observed. Reaction progress wasmonitored by anal. RP-HPLC and after 2 h all doxorubicin had reacted.The mixture was then diluted with H₂O (1.5 mL), cooled to 4° C. andcentrifuged. The supernatant was decanted. The residual pellet wasredissolved in DMF (1 mL) and diluted with 0.1% aq TFA (2 mL). Thissolution was applied to a solid-phase extraction cartridge (MerckLiChrolut RP-18, 500 mg; preconditioned successively with MeOH and 0.1%aq TFA); the cartridge was washed with 0.1% aq TFA (4 mL) and elutedwith 6:4 MeCN/H₂O (containing 0.1% TFA) in two fractions (2×4 mL). Thefirst fraction contained the title compound and was used directly in thenext step.

N-{3-[3-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)succinimido]benzoyl}doxorubicin(SEQ ID No. 27)

The above N-[3-(maleimido)benzoyl]doxorubicin solution was diluted withDMF (1 mL) and Et₃N (50 μL) was added. The solution turned dark brown.H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (5 mg), dissolved in DMF (1 mL) was then added. Themixture was stirred and the brown colour was observed to discharge,leaving a light red solution. The reaction was monitored by anal.RP-HPLC. After 1.5 h, all 3-(maleimido-benzoyl)doxorubicin had reacted.The solution was acidified with AcOH (0.5 mL), diluted with water (3 mL)and applied to a pre-conditioned solid-phase extraction cartridge (MerckLiChrolut RP-18, 500 mg). The cartridge was washed with 0.1% aq TFA (6mL) and eluted (6 mL of 6:4 MeCN/water (containing 0.1% TFA)). Theeluate was dried by vacuum centrifugation. The residue was redissolvedin 0.1% aq TFA (2 mL), filtered and purified by preparative RP-HPLC(20-40% MeCN gradient). Peak fractions were collected, monitored(analytical RP-HPLC) and pooled as appropriate. After vacuumcentrifugation, pure title compound (1.2 mg) was obtained. Anal.RP-HPLC: t_(R)=15.6 & 15.8 min (partly resolved thioether diastereomers)(0-60% MeCN gradient; purity>95%, λ=200-300 nm). DE MALDI-TOF MS:[M+H]⁺=3094, [M+2H]²⁺=1548 (C₁₄₅H₂₀₇N₃₇O₃₅S₂=3092.56).

Example 3H-Cys-D-Lys-D-Lys-D-Trp-D-Lys-D-Met-D-Arg-D-Arg-D-Asn-D-Gln-D-Phe-D-Trp-D-Ile-D-Lys-D-Ile-D-Gln-D-Arg-NH₂(SEQ ID No. 29)

Starting from Rink Amide AM resin (0.64 mmol/g; Novabiochem), thesequenceH-Cys(Trt)-D-Lys(Boc)-D-Lys(Boc)-D-Trp-D-Lys(Boc)-D-Met-D-Arg(Pmc)-D-Arg(Pmc)-D-Asn(Trt)-D-Gln(Trt)-D-Phe-D-Trp-D-Ile-D-Lys(Boc)-D-Ile-D-Gln(Trt)-D-Arg(Pmc)-resin(SEQ ID No. 29) was assembled in quantitative yield. The peptidyl resinwas cleaved/deprotected (10 mL cleavage reagent/g; 2 h) and the crudepeptide was isolated by precipitation from Et₂O,centrifugation/decantation and drying. An aliquot (100 mg) of thismaterial was redissolved in 0.1% aq TEA (2 mL) and filtered. Theresulting solution was purified by preparative RP-HPLC (17.5-27.5% MeCNgradient) to afford, after vacuum centrifugation, pure title compound(36.4 mg). Anal. RP-HPLC: t_(R)=16.3 min (17.5-27.5% MeCN gradient;purity>99%, λ=214 nm). DE MALDI-TOF MS: [M+H]⁺=2350.1(C₁₀₇H₁₇₄N₃₆O₂₀S₂=2348.89).

N-{3-[3-(H-Cys-D-Lys-D-Lys-D-Trp-D-Lys-D-Met-D-Arg-D-Arg-D-Asn-D-Gln-D-Phe-D-Trp-D-Ile-D-Lys-D-Ile-D-Gln-D-Arg-NH₂)succinimido]benzoyl}doxorubicin(SEQ ID No. 29)

N-[3-(Maleimido)benzoyl]doxorubicin (12.6 mg, 17 μmol) andH-Cys-D-Lys-D-Lys-D-Trp-D-Lys-D-Met-D-Arg-D-Arg-D-Asn-D-Gln-D-Phe-D-Trp-D-Ile-D-Lys-D-Ile-D-Gln-D-Arg-NH₂(SEQ ID No. 29) (20 mg, 8.5 μmol) were dissolved in DMF (1 mL) and Et₃N(100 μL) was added. The mixture was stirred for 2 h, quenched byaddition of AcOH (0.5 mL), diluted with water (0.5 mL) and filtered. Thefiltrate was purified by preparative RP-HPLC (20-40% MeCN gradient) toafford the pure title compound as a red solid (6.3 mg). Anal. RP-HPLC:t_(R)=16.3 min (0-60% MeCN gradient; purity>95%). DE MALDI-TOF MS:[M+H]⁺=3092.7, (C₁₄₅H₂₀₈N₃₈O₃₄S₂=3091.57).

Example 4 2′-(Maleimidopropionoyl)paclitaxel

A mixture of paclitaxel (29.2 μmol, 25 mg), 3-maleimidopropionic acid(0.120 mmol, 20.3 mg) and DIC (66 μmol, 10.3 μL) in pyridine (1 mL) wasstirred for 1 hr. The solvent was evaporated, the residue was treatedwith water and extracted with CH₂Cl₂. The organic phase was washed withwater and brine and was dried over MgSO₄, The solvent was evaporated todryness to afford 22.2 mg (76%) colourless solid, which wasrecrystallised from EtOAc/hexane to provide the pure title compound.¹H-NMR (300 MHz, CDCl₃) δ: 1.13, 1.22, 1.68, 1.91 (s, each 3H, CH₃),2.23, 2.47 (s, each 3H, Ac—CH₃), 2.35 (m, 2H, H6), 2.78 (t, 4H, J=5.40Hz, CH₂), 2.84 (m, 2H, H14), 3.81 (m, 2H, CH₂), 3.87 (m, 1H, H3), 4.26(m, 2H, H20), 4.44 (dd, 1H, J=10.87, 4.25 Hz, H7), 4.98 (d, 1H, J=7.69Hz, H5), 5.47 (d, 1H, J=3.45 Hz, H2′), 5.68 (d, 1H, J=7.09 Hz, H3′),6.05 (dd, 1H, J=9.28, 5.86 Hz, H2), 6.28 (s, 1H, H10), 6.18 (t, 1H,J=8.77 Hz, H13), 6.49 (s, 2H, CH═CH), 8.16-7.34 (m, 15H, Ph). ¹³C-NMR(75 MHz; CDCl₃) δ: 10.01, 15.20, 21.22, 22.54 23.09, 27.18, 32.90,33.71, 35.90, 43.54, 45.96, 52.86, 58.89, 72.18, 72.53, 74.86, 75.51,76.02, 79.52, 81.42, 84.89, 126.94, 127.91, 128.74, 128.94, 129.14,129.45, 129.59, 130.65, 132.39, 133.11, 133.85, 134.09, 134.46, 137.17,143.25, 167.45, 168.01, 168.10, 169.77, 170.29, 171.10, 171.69, 204.24.

2′-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]paclitaxel(SEQ ID No. 27)

A solution of 2′-(maleimidopropionoyl)paclitaxel (10 μmol, 10.05 mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (10 μmol, 23.5 mg) in DMF (1 mL) was added Et₃N (1.39μL, 10 μmol). The reaction mixture was stirred for 1 h. It was dilutedwith 0.1% aq TFA (0.5 mL), filtered and purified by preparative RP-HPLC(10-70% MeCN gradient). Pure title compound (20.5 mg, 62%) was obtainedas a colourless solid. Anal. RP-HPLC: t_(R)=17.4 min (0-60% MeCNgradient, purity>97%). DE MALDI-TOF MS: [M+H]⁺=3355.9(C₁₆₁H₂₂₉N₃₇O₃₈S₂=3354.90).

Example 54(5)-carboxyfluorescein-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂(SEQ ID No. 18)

The peptide sequence was assembled on Rink Amide AM resin (0.65 mmol/g,385 mg; Novabiochem) to affordH-βAla-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-Gly-Cys(Trt)-Gly-resin(SEQ ID No. 18) (1.50 g, quant.). An aliquot of this peptidyl resin (450mg, 75 μmol) was stirred for 18 h in the dark with a solution of4(5)-carboxyfluorescein (113 mg, 0.3 mmol), PyBOP (156 mg, 0.3 mmol),HOBt (41 mg, 0.3 mmol) and DIEA (78 μL, 0.45 mmol) in DMF (4 mL). Resinwas collected on a sinter and washed successively with DMF, CH₂Cl₂ andEt₂O. After drying, the resin was treated with cleavage reagent (5 mL,1.5 h) in the dark. The product was isolated by precipitation with Et₂Oand centrifugation (237 mg yellow powder). An aliquot (100 mg) waspurified by preparative RP-HPLC (22.5-32.5% MeCN gradient) to afford thepure title compound (36.9 mg) as a yellow film after isolation by vacuumcentrifugation. Anal. RP-HPLC: t_(R)=18.6 & 19.2 min (resolved 4- and5-carboxyfluorescein geometric isomers) (22.5-32.5% MeCN gradient,purity>99%, λ=214 nm). DE MALDI-TOF MS: [M+H]⁺=2892.2, [M+Na]⁺=2913.7(C₁₃₅H₁₉₅N₃₉O₂₉S₂=2892.4).

2′-[Succinimidopropionoyl-(4(5)-carboxyfluorescein-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂)]paclitaxel(SEQ ID No. 18)

To a solution of 2′-(maleimidopropionoyl)paclitaxel (12.3 μmol, 12.4 mg)and4(5)-carboxyfluorescein-bAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂(SEQ ID No. 18) (4.3 μmol, 12.5 mg) in DMF (1 mL) was added Et₃N (1.8μL). The reaction mixture was stirred for 1 h. It was diluted with 0.1%aq TFA (0.5 mL), filtered and purified by RP-HPLC (10-70% MeCN gradient)to afford pure title compound (3.2 mg) as a colourless solid. Anal.RP-HPLC: t_(R)=21.6 min (0-60% MeCN gradient, purity>97%). DE MALDI-TOFMS: [M+H]⁺=3397.35 (C₁₈₉H₂₅₁N₄₁O₄₆S₂=3397.40).

Example 6H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂(SEQ ID No. 27)

Starting from Rink Amide AM resin (0.69 mmol/g, Novabiochem),H-Cys(Trt)-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-resin(SEQ ID No. 27) was assembled. After deprotection (1.5 h), the crudepeptide was obtained by precipitation from Et₂O,centrifugation/decantation, and drying. Aliquots (total 472 mg) werepurified by preparative RP-HPLC (16.5-26.5% MeCN gradient) to afford thepure title compound (109.9 mg). Anal. RP-HPLC: t_(R)=16.0 min(17.5-27.5% MeCN gradient, purity>99%, λ=214 nm). DE MALDI-TOF MS:[M+H]⁺=2349.3 (C₁₀₇H₁₇₄N₃₆O₂₀S₂=2348.89).

2′-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]paclitaxel(SEQ ID No. 27)

To a solution of 2′-(maleimidopropionoyl)paclitaxel (9 μmol, 9 mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂(SEQ ID No. 27) (9 μmol, 20.9 mg) in DMF (1 mL) was added Et₃N (1.8 μL).The mixture was stirred for 1 h, diluted with 0.1% aq TFA (0.5 mL),filtered and purified by preparative RP-HPLC (10-70% MeCN gradient). Thepure title compound (15.9 mg, 53%) was obtained as a colourless solid.Anal. RP-HPLC: t_(R)=18.5 min (0-60% MeCN gradient, purity>97%). DEMALDI-TOF MS: [M+H]⁺=3353.6 (C₁₆₁H₂₃₀N₃₈O₃₇S₂=3353.91).

Example 7 H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ (SEQ ID No. 19)

Starting from Rink Amide AM resin (0.69 mmol/g, Novabiochem),H-Cys(Trt)-βAla-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-resin(SEQ ID No. 19) was assembled. After deprotection (1.5 h), the crudepeptide was obtained by precipitation from Et₂O,centrifugation/decantation, and drying. Aliquots (total 246 mg) werepurified by preparative RP-HPLC (6.5-16.5% MeCN gradient) to afford thepure title compound (106.4 mg). Anal. RP-HPLC: t_(R)=15.8 min (6.5-16.5%MeCN gradient, purity>95%, λ=214 nm). DE MALDI-TOF MS: [M+H]⁺=1205.4(C₅₂H₉₂N₂₀O₉S₂=1205.55).

2′-[Succinimidopropionoyl-(H-Cys-bAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]paclitaxel(SEQ ID No. 19)

To a solution of 2′-(maleimidopropionoyl)paclitaxel (17 μmol, 17.4 mg)and H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ (SEQ ID No. 19) (15 μmol,18.1 mg) in DMF (1 mL) was added Et₃N (2.0 μL). The mixture was stirredfor 1 h, filtered and purified by preparative RP-HPLC (10-70% MeCNgradient). The pure title compound (9.4 mg) was obtained as a colourlesssolid. Anal. RP-HPLC: t_(R)=17.2 min (0-60% MeCN gradient, purity>97%).DE MALDI-TOF MS: [M+H]⁺=2211.7 (C₁₀₆H₁₄₈N₂₂O₂₆S₂=2210.57.

Example 8 2′-Methoxyacetyl-7-maleimidopropionoyl)paclitaxel

A solution of paclitaxel (29 μmol, 25 mg), methoxyacetic acidN-hydroxysuccinimidyl ester (0.176 mmol, 32.8 mg) and of DIEA (0.176mmol, 30.6 μL) in CH₂Cl₂ (1 mL) was heated under reflux for 4 h.Methanol (1.6 μL) was added. After stirring for 10 min, the reactionmixture was washed with 0.1 M aq HCl., water, brine, and was dried onMgSO₄. The solvent was evaporated in vacuo to afford2′-(methoxyacetyl)paclitaxel as a white solid (24.8 mg, 91%). Thismaterial (30 μmol), together with 3-maleimidopropionic acid, DIC (14.1μL, 90 mmol) and DMAP (20 μmol, 2.6 mg) was dissolved in CH₂Cl₂ (2.5 mL)and the mixture was stirred for 40 min. It was washed with water anddried on MgSO₄. The solvent was removed in vacuo to afford alight-yellow solid. This was redissolved in DMF/MeOH, filtered andpurified by preparative RP-HPLC (20-70% MeCN gradient) to afford thepure title compounds as a colourless solid (24.4 mg, 76%). Anal.RP-HPLC: t_(R)=21.8 min (10-70% MeCN gradient, purity>98%). ¹H-NMR (300MHz, CDCl₃) δ: 1.15, 1.20, 1.79, 1.96 (s, each 3H, CH₃x4), 2.20, 2.45(s, each 3H, Ac—CH₃x2), 2.34 (m, 2H, H6), 2.63 (m, 4H, H14, CH₂), 3.40(s, 3H, OCH₃), 3.73-3.94 (m, 3H, CH₂, H3), 4.16-4.21 (m, 2H, H20), 4.97(d, 1H, J=8.06 Hz, H5), 5.54-5.69 (m, 3H, H7, H2, H3′), 5.98 (m, 1H,H2′), 6.22 (s, 1H, H10), 6.24 (m, 1H, H13), 6.68 (s, 2H, CH═CH),7.12-8.13 (m, 15H, Ph).

2′-Methoxyacetyl-7-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]paclitaxel(SEQ ID) No. 27)

To a solution of 2′-methoxyacetyl-7-(maleimidopropionoyl)paclitaxel (11μmol, 12.3 mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (11 μmol, 26.8 mg) in DMF (1 mL) was added Et₃N (1.58μL, 11 μmol). The mixture was stirred for 2 h, diluted with 0.1% aq TFA(0.5 mL) and purified by preparative RP-HPLC (10-70% MeCN gradient). Thepure title compound was obtained as a colourless solid (15.5 mg, 40%).Anal. RP-HPLC: t_(R)=15.1 min (10-70% MeCN gradient, purity>97%). DEMALDI-TOF MS: [M+H]⁺=3425.99 (C₁₆₄H₂₃₃N₃₇O₄₀S₂=3424.96).

7-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]paclitaxel(SEQ ID No. 27)

To2′-methoxyacetyl-7-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]paclitaxel(SEQ ID No. 27) (35 μmol, 11.9 mg) in MeOH (1 mL) was added ethanolamine(0.21 μL). The mixture was stirred for 1 h, diluted with 0.1% aq TFA(0.5 mL), filtered and purified by preparative RP-HPLC (10-70% MeCNgradient). The pure title compound was obtained as a colourless solid(5.6 mg, 48%). Anal. RP-HPLC: t_(R)=14.3 min (10-70% MeCN gradient,purity>97%). DE MALDI-TOF MS: [M+H]⁺=3355.7, (C₁₆₁H₂₂₉N₃₇O₃₈S₂=3354.90).

Example 9 2′-(p-Methoxytrityl)paclitaxel

A solution of paclitaxel (0.632 mmol, 540 mg) and p-methoxytritylchloride (10 mol eq) in CH₂Cl₂ (10 mL) was treated with pyridine (1.3mL) under N₂. After stirring of the mixture for 22 h, solvents wereevaporated in vacuo. The residue was redissolved in EtOAc, washed withwater and brine and was dried on MgSO₄. The solvent was evaporated toafford a light yellow solid which was purified by flash chromatography(8:9 EtOAc/PE) to afford the pure title compound in a quantitativeyield. Recrystallisation from EtOAc/CH₂Cl₂ gave light yellow crystals.¹H-NMR (300 MHz, CDCl₃) δ: 1.08, 1.15, 1.51, 1.65, (s, each 3H, CH₃x4),1.90 (m, 1H, H6), 2.25, 2.29 (s, each 3H, Ac—CH₃x2), 2.55 (m, 1H, H6),2.54 (m, 2H, H14), 3.75 (s, 3H, OCH₃), 3.66 (m, 1H, H3), 4.20 (m, 2H,H20), 4.40 (m, 1H, H7), 4.62 (m, 1H, H2′), 4.94 (d, 1H, J=8.06 Hz, H5),5.61 (m, 1H, H2), 5.70 (m, 2H, H13, H3′), 6.19 (s, 1H, H10), 6.72-8.08(m, 29H, Ph).

2′-(p-Methoxytrityl)-7-(maleimidopropionoyl)paclitaxel

2′-(p-Methoxytrityl)paclitaxel (35 μmol, 38.4 mg) and pyridine (125 μL)were dissolved in CH₂Cl₂ (2 mL). A solution of 3-maleimidopropionic acid(1.48 mmol, 250.5 mg), DIC (0.80 mmol, 101.5 mg) and DMAP (10 mg) inCH₂Cl₂ (2 mL) was added and the mixture was stirred for 1 h. The solventwas evaporated and the residue was partitioned between water and CH₂Cl₂.The organic layer was washed with water, brine and was dried on MgSO₄.The solvent was removed and the residue was purified by Chromatotron®centrifugal thin-layer chromatography (5:4 EtOAc/PE). Recrystallisationfrom EtOAc/CH₂Cl₂ afforded the title compound as a colourless solid (22mg, 49%). ¹H-NMR (300 MHz, CDCl₃) δ: 1.18, 1.12, 1.76, 1.96 (s, each 3H,CH₃x4), 2.17, 2.26 (s, each 3H, Ac—CH₃x2), 2.10, 2.34 (m, 2H, H6), 2.62(m, 4H, H14, CH₂-Mim), 3.75 (s, 3H, OCH₃), 3.73-3.79 (m, 3H, CH₂-Mim,H3), 4.06 (m, 2H, H20), 4.61 (d, 1H, J=3.47 Hz, H2′), 4.76 (d, 1H,J=9.52 Hz, H5), 5.53 (m, 1H, H7), 5.60 (d, 1H, J=6.98 Hz, H3′), 5.71 (m,1H, H2), 6.14 (s, 1H, H10), 6.60 (m, 3H, H13, CH═CH), 6.75-7.79 (m, 29H,Ph).

7-(Maleimidopropionoyl)paclitaxel

A solution of 2′-p-methoxytrityl)-7-(maleimidopropionoyl)paclitaxel (17μmol, 22 mg), anisole (1.72 mmol, 186.4 mg) and chloroacetic acid (0.172mmol, 16.3 mg) in CH₂Cl₂ (10 mL ) was stirred for 4 h. The reactionmixture was washed with 1% aq Na₂CO₃, water, brine and was dried onMgSO₄. The solvent was evaporated to dryness and the residue waspurified by Chromatotron®& centrifugal thin-layer chromatography (1:1EtOAc/PE) to afford pure title compound as a white solid (24 mg), whichwas recrystallised from EtOAc/CH₂Cl₂. ¹H-NMR (300 MHz, CDCl₃) δ: 1.15,1.18, 1.20, 1.76, 2.04 (s, each 3H, CH₃x4), 2.18, 2.37 (s, each 3H,Ac—CH₃x2), 2.34 (m, 2H, H6), 2.64 (m, 4H, H14, CH₂), 3.78-3.91 (m, 3H,CH₂, H3), 4.12 (m, 2H, H20), 4.71 (d, 1H, J=3.25 Hz, H2′), 4.94 (d, 1H,J=8.17 Hz, H5), 5.54 (dd, 1H, J=10.46, 7.21 Hz, H7), 5.66 (d, 1H, J=6.88Hz, H3′), 5.80 (dd, 1H, J=8.92, 2.42 Hz, H2), 6.15 (m, 1H, H13), 6.18(s, 1H, H10), 6.68 (s, 2H, CH═CH), 7.10-8.12 (m, 15H, Ph).

7-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]paclitaxel(SEQ ID No. 27)

To a solution of 7-(maleimidopropionoyl)paclitaxel (4.8 μmol, 4.8 mg)andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (4.8 μmol, 11.2 mg) in DMF (1 mL) was added Et₃N (0.67μL). The mixture was stirred for 30 min, filtered and purified bypreparative RP-HPLC (10-70% MeCN gradient) to afford the pure titlecompound as a colourless solid (8.6 mg, 54%). Anal. RP-HPLC: t_(R)=14.3min (10-70% MeCN gradient, purity>97%). DE MALDI-TOF MS: [M+H]⁺=3355.0(C₁₆₁H₂₂₉N₃₇O₃₈S₂=3354.90).

Example 10 4-(Maleimidopropionoyl)podophyllotoxin

A solution of podophyllotoxin (60 μmol, 25.6 mg), 3-maleimidopropionicacid (0.31 mmol, 52.4 mg), DIC (0.17 mmol, 21.5 mg) and DMAP (80 μmol,10 mg) in CH₂Cl₂ (2 mL) was stirred for 1 h. The solvent was evaporatedin vacuo and the residue was redissolved in DMF/MeOH (1 mL) and purifiedby preparative RP-HPLC (20-70% MeCN gradient) to afford the pure titlecompound as a colourless solid (7.3 mg). Anal. RP-HPLC: t_(R)=20.1 min(0-60% MeCN gradient, purity>95%). ¹H-NMR (300 MHz, CDCl₃) δ: 2.66-2.71(t, J=6.3 Hz, 2H, CH₂), 2.82-2.84 (m, 2H, H2 and H3), 3.69 (s, 6H,OCH₃x2), 3.75 (s, 3H, OCH₃), 3.83 (t, J)=6.3 Hz, 2H, CH₂), 4.12 (t,J=9.92 Hz, 1H, H11), 4.31 (m, 1H, H11), 4.53 (d, J=11.4 Hz, 1H, H1),5.80 (d, J=8.7 Hz, 1H, H4), 5.92 (dd, J=5.49, 1.17 Hz, 2H, OCH₂O), 6.32(s, 2H, H2′6′), 6.47 (s, 1H, H8), 6.66 (s, 2H, CH═CH), 6.74 (s, 1H, H5).

4-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ill-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]podophyllotoxin(SEQ ID No. 27)

To a solution of 4-(maleimidopropionoyl)podophyllotoxin (8 μmol, 5 mg)andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (7.7 μmol, 18 mg) in 1 DMF (1 mL) was added Et₃N (1.06μL, 11.4 μmol). The mixture was stirred for 30 min, diluted with 0.1% aqTFA (0.5 mL), filtered and purified by preparative RP-HPLC (10-60% MeCNgradient) to afford the pure title compound as a colourless solid (7.8mg, 35%). Anal. RP-HPLC: t^(R)=12.8 min (0-60% MeCN gradient,purity>97%). DE MALDI-TOF MS: [M+H]⁺=2915.34 (C₁₃₆H₂₀₀N₃₆O₃₂S₂=2915.40).

Example 11Biotinamidocaproyl-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂(SEQ ID No. 18)

H-βAla-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-Gly-Cys(Trt)-Gly-resin(SEQ ID No. 18) (450 mg, 75 μmol) was stirred with a solution ofbiotinamidocaproic acid N-hydroxysuccinimidyl ester (136 mg, 0.3 mmol),HOBt (41 mg, 0.3 mmol) and DIEA (105 μL, 0.6 mmol) in DMF (3 mL) for 18h. The peptidyl resin was collected on a sinter and washed successivelywith DMF, CH₂Cl₂, and Et₂O. After drying in vacuo, it was treated withcleavage reagent (5 mL, 1.5 h). The biotinylated peptide was isolated byprecipitation with Et₂O and centrifugation (244 mg product). An aliquot(120 mg) was purified by preparative RP-HPLC (20-30% MeCN gradient) toafford the pure title compound as a colourless solid (63.8 mg). Anal.RP-HPLC: t_(R)=16.7 min (20-30% MeCN gradient, purity>99%, λ=214 nm). DEMALDI-TOF MS: [M+H]⁺=2874.3, [2M+H]⁺=5738.7, [M+2H]²⁺=1437.8(C₁₃₀H₂₁₀N₄₂O₂₆S₃=2873.52).

4-[Succinimidopropionoyl-(biotinamidocaproyl-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂)]podophyllotoxin(SEQ ID No. 18)

To a solution of 4-(maleimidopropionoyl)podophyllotoxin (7 μmol, 4 mg)andbiotinamidocaproyl-bAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂(SEQ ID No. 18) (7 μmol, 20.7 mg) in DMF (0.5 mL) was added Et₃N (1.0μL). The mixture was stirred for 1 h, diluted with 0.1% aq TFA (0.5 mL),filtered and purified by preparative RP-HPLC (10-70% MeCN gradient). Thepure title compound was obtained as a colourless solid (2.2 mg). Anal.RP-HPLC: t_(R)=17.2 min (0-60% MeCN gradient, purity>97%). DE MALDI-TOFMS: [M+H]⁺=3438.9 (C₁₅₉H₂₃₇N₄₃O₃₇S²=3439.05).

Example 124-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]podophyllotoxin(SEQ ID No. 27)

To a solution or 4-maleimidopropionoyl)podophyllotoxin (20 μmol, 12.2mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂(SEQ ID No. 27) (15 μmol, 34.7 mg) in DMF (1.5 mL) was added Et₃N (5μL). The mixture was stirred for 40 min and purified by preparativeRP-HPLC (0-60% MeCN gradient) to afford the pure title compound as acolourless solid (30.1 mg, 69%). Anal. RP-HPLC: t_(R)=15.8 min (0-60%MeCN gradient, purity>98%). DE MALDI-TOF MS: [M+H]⁺=2914.4(C₁₃₆H₂₀₁N₃₇O₃₁S₂=2914.41).

Example 13H-Cys-D-Arg-D-Gln-D-Ile-D-Lys-D-Ile-D-Trp-D-Phe-D-Gln-D-Asn-D-Arg-D-Arg-D-Nle-D-Lys-D-Trp-D-Lys-D-Lys-NH₂(SEQ ID No. 27)

Starting from Rink Amide AM resin (0.69 mmol/g, Novabiochem),H-Cys(Trt)-D-Arg(Pmc)-D-Gln(Trt)-D-Ile-D-Lys(Boc)-D-Ile-D-Trp-D-Phe-D-Gln(Trt)-D-Asn(Trt)-D-Arg(Pmc)-D-Arg(Pmc)-D-Nle-D-Lys(Boc)-D-Trp-D-Lys(Boc)-D-Lys(Boc)-resin(SEQ ID No. 27) was assembled. After deprotection (1.5 h), the crudepeptide was obtained by precipitation from Et₂O,centrifugation/decantation, and drying. Aliquots (total 246 mg) werepurified by preparative RP-HPLC (17.5-27.5% MeCN gradient) to afford thepure title compound (45.9 mg). Anal. RP-HPLC: t_(R)=16.9 min (17.5-27.5%MeCN gradient, purity>99%, I=214 nm). DE MALDI-TOF MS: [M+H]⁺=2330.3(C₁₀₈H₁₇₆N₃₆O₂₀S=2330.85).

4-[Succinimidopropionoyl-(H-Cys-D-Arg-D-Gln-D-Ile-D-Lys-D-Ile-D-Trp-D-Phe-D-Gln-D-Asn-D-Arg-D-Arg-D-Nle-D-Lys-D-Trp-D-Lys-D-Lys-NH₂)]podophyllotoxin(SEQ ID No. 27)

To a solution of 4-(maleimidopropionoyl)podophyllotoxin (11 μmol, 6.2mg) andH-Cys-D-Arg-D-Gln-D-Ile-D-Lys-D-Ile-D-Trp-D-Phe-D-Gln-D-Asn-D-Arg-D-Arg-D-Nle-D-Lys-D-Trp-D-Lys-D-Lys-NH₂(SEQ ID No. 27) (7 μmol, 17 mg) in DMF (1 mL) was added Et₃N (1.4 μL).The mixture was stirred for 30 min, filtered and purified by preparativeRP-HPLC (0-60% MeCN gradient) to afford the pure title compound as acolourless solid (10.5 mg, 52%). Anal. RP-HPLC: t^(R)=15.8 min (0-60%MeCN gradient, purity>98%). DE MALDI-TOF MS: [M+H]⁺=2895.66(C₁₃₇H₂₀₃N₃₇O₃₁S₂=2896.37).

Example 144-[Succinimidopropionoyl-CH-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]podophyllotoxin(SEQ ID No. 19)

To a solution of 4-(maleimidopropionoyl)podophyllotoxin (17.7 μmol, 10mg) and H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ (SEQ ID No. 19) (25μmol, 30.4 mg) in DMF (1.5 mL) was added Et₃N (3.5 μL). The mixture wasstirred for 40 min, filtered and purified by preparative RP-HPLC (0-60%MeCN gradient). The pure title compound was obtained as a colourlesssolid (17.8 mg, 57%). Anal. RP-HPLC: t_(R)=14.8 min (0-60% MeCNgradient, purity>98%). DE MALDI-TOF MS: [M+H]⁺=1772.3(C₈₁H₁₁₉N₂₁O₂₀S₂=1771.07).

Example 15 H-Cys-βAla-D-Arg-D-Arg-D-Met-D-Lys-D-Trp-D-Lys-D-Lys-NH₂ (SEQID No. 19)

Starting from Rink Amide AM resin (0.69 mmol/g, Novabiochem),H-Cys(Trt)-βAla-D-Arg(Pmc)-D-Arg(Pmc)-D-Met-D-Lys(Boc)-D-Trp-D-Lys(Boc)-D-Lys(Boc)-resin(SEQ ID No. 19) was assembled. After deprotection (1.5 h), the crudepeptide was obtained by precipitation from Et₂O,centrifugation/decantation, and drying. Aliquots (total 237 mg) werepurified by preparative RP-HPLC (8-18% MeCN gradient) to afford the puretitle compound (66 mg). Anal. RP-HPLC: t_(R)=12.9 min (9-19% MeCNgradient, purity>99%, λ=214 nm). DE MALDI-TOF MS: [M+H]⁺=1207.2(C₅₂H₉₂N₂₀O₉S₂=1205.55).

4-[Succinimidopropionoyl-(H-Cys-b-Ala-D-Arg-D-Arg-D-Met-D-Lys-D-Trp-D-Lys-D-Lys-NH₂)]podophyllotoxin(SEQ ID No. 19)

To a solution of 4-(maleimidopropionoyl)podophyllotoxin (18.9 μmol, 10.7mg) and H-Cys-βAla-D-Arg-D-Arg-D-Met-D-Lys-D-Trp-D-Lys-D-Lys-NH₂ (SEQ IDNo. 19) (28 μmol, 33.8 mg) in DMF (1.5 mL) was added Et₃N (1.5 μL). Themixture was stirred for 40 min, filtered and purified by preparativeRP-HPLC (0-60% MeCN gradient). The pure title compound was obtained as acolourless solid (6.9 mg, 21%). Anal. RP-HPLC: t_(R)=14.8 min (0-60%MeCN gradient, purity>98%). DE MALDI-TOF MS: [M+H]⁺=1771.5(C₈₁H₁₁₉N₂₁O₂₀S₂=1771.07).

Example 16 4′-(Maleimidopropionoyl)epipodophyllotoxin

A solution of 4′-demethylepipodophyllotoxin (12 mmol, 5 mg),3-maleimidopropionic acid (50 μmol, 12.2 mg) and DIC (28 μmol, 3.47 mg)in pyridine (1 mL) was stirred for 30 min. MeOH (0.5 mL) was added andthe mixture was purified by preparative RP-HPLC (0-60% MeCN gradient) toafford the pure title compound as a colourless solid (4.2 mg, 62%).Anal. RP-HPLC: t_(R)=17.6 min (0-60% MeCN gradient, purity>95%). ¹H-NMR(300 MHz, CDCl₃) δ: 2.84 (m, 1H, H3), 2.99 (t, J=7.44 Hz, 2H, CH₂-Mim),3.32 (dd, J=14.04, 5.07 Hz, 1H, H2), 3.69 (s, 6H, OCH₃x2), 3.95 (t,J=7.44 Hz, 2H, CH₂-Mim), 4.39 (dd, J=8.13, 4.28 Hz, 2H, H11), 4.66 (d,J=5.00 Hz, 1H, H1), 4.89 (d, J=3.32 Hz, 1H, H4), 6.01 (d, J=6.42 Hz, 2H,OCH₂O), 6.32 (s, 2H, H2′6′), 6.57 (s, 1H, H8), 6.74 (s, 2H, CH═CH), 6.90(s, 1H, H5). ¹³C-NMR (75 MHz, CDCl₃) δ: 28.64, 31.02, 32.55, 37.33,39.53, 42.99, 55.15, 65.78, 66.56, 100.65, 106.54, 107.97, 109.65,130.68, 130.92, 133.21, 136.96, 146.62, 147.61, 150.39, 167.36, 169.30,173.89.

4′-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]epipodophyllotoxin(SEQ ID No. 27)

To a solution of 4′-(maleimidopropionoyl)epipodophyllotoxin (2.3 μmol,1.3 mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (2.3 μmol, 5.4 mg) in DMF (0.5 mL) was added Et₃N (0.21μL, 2.3 μmol). The mixture was stirred for 40 min, diluted with 0.1% aqTFA (1 mL), filtered and purified by preparative RP-HPLC (0-60% MeCNgradient) to afford the pure title compound as a colourless solid (3.2mg, 48%). Anal. RP-HPLC: t_(R)=14.6 min (0-60% MeCN gradient,purity>98%). DE MALDI-TOF MS: [M+H]⁺=2902.2 (C₁₃₅H₁₉₈N₃₆O₃₂S₂=2901.37).

Example 174′-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]epipodophyllotoxin(SEQ ID No. 27)

To a solution of 4′-(maleimidopropionoyl)epipodophyllotoxin (7 μmol, 4mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂(SEQ ID No. 27) (6 μmol, 15 mg) in DMF (0.5 mL) was added Et₃N (1 μL).The mixture was stirred for 40 min and was purified by preparativeRP-HPLC (0-60% MeCN gradient) to afford the pure title compound as acolourless solid (14.1 mg, 81%). Anal. RP-HPLC: t_(R)=19.7 min (0-60%MeCN gradient, purity>98%). DE MALDI-TOF MS: [M+H]⁺=2900.4,C₁₃₅H₁₉₉N₃₇O₃₁S₂=2900.39.

Example 184′-[Succinimidopropionoyl-(H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]epipodophyllotoxin(SEQ ID No. 19)

To a solution of 4′-(maleimidopropionoyl)epipodophyllotoxin (14 μmol,7.9 mg) and H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ (SEQ ID No. 19)(26 μmol, 31.5 mg) in DMF (1 mL) was added Et₃N (1.9 μL). After stirringfor 40 min, the mixture was purified by preparative RP-HPLC (0-60%gradient) to afford the pure title compound as a colourless solid (15.8mg, 63%). Anal. RP-HPLC: t_(R)=13.3 min (0-60% MeCN gradient,purity>98%). DE MALDI-TOF MS: [M+H]⁺=1757.2 (C₈₀H₁₁₇N₂₁O₂₀S₂=1757.05).

Example 19 4′-(Chloroacetyl)epipodophyllotoxin

To a stirring solution of 4′demethylepipodophyllotoxin (0.50 mmol, 200mg) and pyridine (40 μL) in CH₂Cl₂ (2 mL) at 0° C., chloroacetylchloride (0.50 mmol, 56.5 mg) was added dropwise. By anal. RP-HPLC about60% of 4′-demethylepipodophyllotoxin starting material was consumedafter 1 hr stirring at 0° C. The reaction mixture was poured intochilled water and this was extracted with CH₂Cl₂. The organic layer waswashed with water, brine and was dried on MgSO4. The solvent wasevaporated in vacuo and the residue was purified by flash chromatography(5:4-3:2 EtOAc/PE). The pure title compound was obtained afterrecrystallisation from EtOAc/PE as a colourless solid (81.5 mg, 34%).¹H-NMR (300 MHz, CDCl₃) δ: 2.78 (m, 1H, H3), 3.25 (dd, J=14.12, 5.07 Hz,1H, H2), 3.68 (s, 6H, OCH₃x2), 4.30 (m, 2H, H11), 4.35 (s, 2H, CH2Cl),4.57 (d, J=5.12 Hz, 1H, H1), 4.83 (d, J=3.37 Hz, H4), 5.96 (d, J=4.10Hz, 2H, OCH₂O), 6.32 (s, 2H, H2′6′), 6.50 (s, 1H, H8), 6.87 (s, 1H, H5).

4′-Chloroacetyl-4-maleimidopropionoyl)epipodophyllotoxin

A solution of 4′-(chloroacetyl)epipodophyllotoxin (0.17 mmol, 81.5 mg),3-maleimidopropionic acid (0.68 mmol, 115.6 mg), DIC (0.376 mmol, 47.5mg), DMAP (73 μmol, 9 mg) and pyridine (20 μL) in CH₂Cl₂ (2 mL) wasstirred for 1 h. The solvent was evaporated to dryness. The resultinglight-yellow solid was redissolved in DMF (1 mL) and was purified bypreparative RP-HPLC (30-70% MeCN gradient) to afford the pure titlecompound as a colourless solid (54.3 mg, 51%). Anal. RP-HPLC: t_(R)=22.0min (0-60% MeCN gradient, purity>97%). ¹H-NMR (300 MHz, CDCl₃) δ: 2.71(t, 2H, J=6.80 Hz, CH₂-Mim), 2.98 (m, 1H, H3), 3.25 (dd, J=14.20, 5.13Hz, 1H, H2), 3.69 (s, 6H, OCH₃x2), 3.87 (t, J=6.83 Hz, 2H, CH₂-Mim),3.88 (m, 1H, H11), 4.33 (s, 2H, CH₂Cl), 4.35 (m, 1H, H11), 4.70 (d,J=5.10 Hz, 1H, H1), 6.01 (d, J=4.23 Hz, 2H, OCH₂O), 6.13 (d, J=3.50 Hz,1H, H4), 6.31 (s, 2H, H2′6′), 6.56 (s, 1H, H8), 6.71 (s, 2H, CH═CH),6.92 (s, 1H, H5).

4′-Chloroacetyl-4-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]epipodophyllotoxin(SEQ ID No. 27)

To a solution of4′-chloroacetyl-4-(maleimidopropionoyl)epipodophyllotoxin (6.8 μmol, 43mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (10 μmol, 25.4 mg) in DMF (1.5 mL) was added Et₃N (2.5μL). The mixture was stirred for 30 min and was purified by preparativeRP-HPLC (10-70% MeCN gradient) to afford the pure title compound as acolourless solid (22 mg, 67%). Anal. RP-HPLC: t_(R)=16.7 min (0-60% MeCNgradient, purity>99%). DE MALDI-TOF MS: [M+H]⁺=2978.3(C₁₃₇H₁₉₉ClN₃₆O₃₃S₂=2977.85).

4′-Demethyl-4-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]epipodophyllotoxin(SEQ ID No. 27)

A solution of4′-chloroacetyl-4-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]epipodophyllotoxin(SEQ ID No. 27) (5.5 μmol, 16.4 mg) in DMF (1 mL) and water (0.5 mL) at0° C. was treated with conc. aq NH₃ solution (20 μL). After 2 min thereaction mixture was acidified by addition of 5% aq AcOH (0.1 mL). Itwas purified by preparative RP-HPLC (0-60% MeCN gradient) to afford thepure title compound as a colourless solid (11.4 mg, 73%). Anal. RP-HPLC:t_(R)=14.9 min (0-60% MeCN gradient, purity>99%). DE MALDI-TOF MS:[M+H]⁺=2902.2 (C₁₃₅H₁₉₈N₃₆O₃₂S₂=2901.37).

Example 20 G2-(Maleimidopropionoyl)etoposide,G3-(maleimidopropionoyl)etoposide, and 4′-(maleimidopropionoyl)etoposide

A solution of etoposide (37.4 μmol, 22 mg), 3-maleimidopropionic acid(78 μmol, 13.2 mg) and DIC (39.6 μmol, 5 mg) in a mixture ofCH₂Cl₂/pyridine (2:0.15) was stirred for 30 min. The solvents wereremoved in vacuo. The resulting light-yellow solid was dissolved in MeOH(1.5 mL) and was purified by preparative RP-HPLC (10-70% MeCN gradient)to afford G2-(maleimidopropionoyl)etoposide (3.4 mg),G3-(maleimidopropionoyl)etoposide (2.4 mg) and4′-(maleimidopropionoyl)etoposide (7.7 mg) as colourless solids (totalyield 48%).

G2-(Maleimidopropionoyl)etoposide

Anal. RP-HPLC: t_(R)=16.7 min (0-60% MeCN gradient, purity>99%). ¹H-NMR(300 MHz, CDCl₃) δ: 1.39 (d, J=4.99 Hz, 3H, CH₃), 2.39 (t, J=7.30 Hz,2H, CH₂-Mim), 2.87 (m, 1H, H3), 3.14 (dd, J=14.20, 5.09 Hz, 1H, H2),3.39 (m, 2H, G4,5), 3.63 (m, 2H, G2,6), 3.72 (m, 2H, CH₂-Mim), 3.76 (s,6H, OCH₃x2), 3.84 (t, J=8.9 Hz, 1H, G3), 4.19 (m, 2H, H11, G6), 4.38 (m,1H, H11), 4.60 (d, J=4.10 Hz, 1H, H4), 4.74-4.84 (m, 2H, H1, G6). 6.00(d, J=5.10 Hz, 2H, OCH₂O), 6.24 (s, 2H, H2′6′), 6.54 (s, 1H, H8), 6.70(s, 2H, CH═CH), 6.75 (s, 1H, H5). ¹³C-NMR (75 MHz, CDCl₃) δ: 20.66,33.48, 33.95, 37.86, 41.46, 44.00, 56.87, 66.75, 68.07, 68.34, 72.15,74.61, 75.26, 80.24, 100.29, 100.434, 102.07, 108.33, 109.00, 111.33,128.75, 130.90, 133.40, 134.50, 134.66, 146.80, 147.27, 149.09, 169.94,170.78, 175.08.

G3-(Maleimidopropionoyl)etoposide

Anal. RP-HPLC: t_(R)=18.4 min (0-60% MeCN gradient, purity>99%). ¹H-NMR(300 MHz, CDCl₃) δ: 1.33 (d, J=5.0 Hz, 3H, CH₃), 2.74 (t, J=7.35 Hz, 2H,CH₂-Mim), 2.91 (m, 1H, H3), 3.28 (dd, J=14.01, 5.26 Hz, 1H, H2), 3.38(m, 2H, G4,5), 3.54 (m, 2H, G2,6), 3.87 (m, 2H, CH₂-Mim), 3.76 (s, 6H,OCH₃x2), 4.16-4.26 (m, 2H, H11, G3), 4.42 (t, J=8.98 Hz, 1H), 4.61 (d,J=5.09 Hz, 1H, H1), 4.68 (m, 1H, G1), 4.91 (d, J=3.34 Hz, H4), 5.13 (m,1H, G3). 6.00 (d, J=11.25 Hz, 2H, OCH₂O), 6.26 (s, 2H, H2′6′), 6.54 (s,1H, H8), 6.71 (s, 2H, CH═CH), 6.83 (s, 1H, H5). ¹³C-NMR (75 MHz, CDCl₃)δ: 0.67, 33.59, 34.13, 37.93, 41.62, 44.11, 56.84, 66.87, 68.26, 68.38,73.39, 74.38, 74.49, 100.17, 102.01, 102.54, 108.25, 109.51, 111.10,128.38, 130.89, 133.28, 134.50, 134.66, 146.84, 147.59, 149.29, 170.64,170.80, 175.38.

4′-(Maleimidopropionoyl)etoposide

Anal. RP-HPLC: t_(R)=17.7 min (0-60% MeCN gradient, purity>99%). ¹H-NMR(300 MHz, CDCl₃) δ: 1.39 (d, J=4.82 Hz, 3H, CH₃), 2.88 (m, 1H, H3), 2.96(t, J=7.24 Hz, 2H, CH₂-Mim, 2.91 (m, 1H, H3), 3.34 (dd, J=14.01, 5.26Hz, 1H, H2), 3.36 (m, 2H, G4,5), 3.45-3.58 (m, 2H, G2,6), 3.92 (t,J=3.20 Hz, 2H, CH₂-Mim), 3.65 (s, 6H, OCH₃x2), 3.76 (m, 1H, G3),4.15-4.27 (m, 2H, H11, G6), 4.43 (m, 1H, H11), 4.62-4.67 (m, 2H, H1,G1), 4.75 (m, 1H, G7), 4.91 (d, J=3.27 Hz, H4), 6.00 (d, J=6.68 Hz, 2H,OCH₂O), 6.25 (s, 2H, H2′6′), 6.54 (s, 1H, H8), 6.71 (s, 2H, CH═CH), 6.82(s, 1H, H5). ¹³C-NMR (75 MHz, CDCl₃) δ: 20.62, 32.41, 33.94, 37.87,41.61, 44.31, 56.52, 66.84, 68.44, 73.47, 74.13, 74.88, 80.06, 100.24,102.10, 102.30, 107.89, 109.36, 111.22, 128.65, 132.68, 134.61, 138.28,147.74, 149.29, 151.76, 168.90, 170.76, 175.56.

G2-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]etoposide(SEQ ID No. 27)

To a solution of G2-(maleimidopropionoyl)etoposide (4.4 μmol, 3.3 mg)andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (5.7 μmol, 13.4 mg) in DMF (0.5 mL) was added Et₃N (0.7μL, 4.9 μmol). The mixture was stirred for 30 min, diluted with 0.1% aqTFA (1 mL) and purified by preparative RP-HPLC (0-60% MeCN gradient) toafford the pure title compound as a colourless solid (10.8 mg, 80%).Anal. RP-HPLC: t_(R)=14.6 min (0-60% MeCN gradient, purity>98%). DEMALDI-TOF MS: [M+H]⁺=3091.1 (C₁₄₃H₂₁₀N₃₆O₃₇S₂=3089.55).

G3-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]etoposide(SEQ ID No. 27)

To a solution of G3-(maleimidopropionoyl)etoposide (3.1 μmol, 2.3 mg)andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (4.3 μmol, 10.2 mg) in DMF (0.5 mL) was added Et₃N (0.6μL, 4.4 μmol). The mixture was stirred for 30 min, diluted with 0.1% aqTFA (1 mL) and purified by preparative RP-HPLC (0-60% MeCN gradient) toafford the pure title compound as a colourless solid (7.4 mg, 79%).Anal. RP-HPLC: t_(R)=14.7 min (0-60% MeCN gradient, purity>98%). DEMALDI-TOF MS: [M+H]⁺=3090.3 (C₁₄₃H₂₁₀N₃₆O₃₇S₂=3089.55).

4′-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]etoposide(SEQ ID No. 27)

To a solution of 4′-(maleimidopropionoyl)etoposide (4.8 μmol, 3.6 mg)andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (5.9 μmol, 13.9 mg) in DMF (0.5 mL) was added Et₃N (0.7μL, 5.1 μmol). The mixture was stirred for 30 min, diluted with 0.1% aqTFA (1 mL) and purified by preparative RP-HPLC (0-60% MeCN gradient) toafford the pure title compound as a colourless solid (11.2 mg, 77%).Anal. RP-HPLC: t_(R)=14.6 min (0-60% MeCN gradient, purity>99). DEMALDI-TOF MS: [M+H]⁺=3090.9 (C₁₄₃H₂₁₀N₃₆O₃₇S₂=3089.55).

Example 21 O-(Maleimidoropionoyl)roscovitine

A solution of roscovitine (29 μmol, 10.3 mg), 3-maleimidopropionic acid(64 μmol, 10.8 mg), DIC (35 μmol, 4.4 mg) and DMAP (2 μmol, 0.35 mg) inof pyridine (1 mL) was stirred for 40 min. The solvent was evaporated invacuo and the resulting light-yellow solid was redissolved in CH₂Cl₂,washed with water and brine and was dried on MgSO₄. The solvent wasevaporated and the title compound was obtained as a light-yellow solid(14.1 mg, 96%). This material was used without further purification inthe next reaction.

O-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]roscovitine(SEQ ID No. 27)

To a solution of O-(maleimidopropionoyl)roscovitine (28 μmol, 14.1 mg)andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (14.9 μmol, 35 mg) in DMF (1.5 mL) was added Et₃N (2 μL,14.5 μmol). The mixture was stirred for 1 h and was purified bypreparative RP-HPLC (10-60% MeCN gradient) to afford the pure titlecompound as a colourless solid (7.2 mg). Anal. RP-HPLC: t_(R)=15.5 min(0-60% MeCN gradient, purity>98%). DE MALDI-TOF MS: [M+H]⁺=2856.1(C₁₃₃H₂₀₄N₄₂O₂₅S₂=2855.44).

Example 22 O-βAla-Bohemine

A solution of bohemine (58.8 μmol, 20 mg), Boc-βAla-OH (0.128 mmol, 24.2mg) DIC (70 μmol, 8.8 mg) and DMAP (9.8 μmol, 1.2 mg) in CH₂Cl₂ (2 mL)was stirred for 2.5 hrs. The solvent was evaporated in vacuo and theresulting white solid was purified by preparative RP-HPLC (10-70% MeCNgradient) to afford O-(Boc-βAla)bohemine as a colourless solid (30 mg).Anal. RP-HPLC: t_(R)=19.8 min (0-60% MeCN gradient, purity>99%). Asolution of O-(Boc-βAla)bohemine (7.6 mg) in 9:1 TFA/water (1 mL) wasstirred for 1 h. The solvent was evaporated to dryness and the residueof title compound was used without further purification in the nextreaction (purity by anal. RP-HPLC was >98%).

O-(βAla-succinyl-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)bohemine(SEQ ID No. 20)

A mixture ofsuccinyl-βAla-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-resin(SEQ ID No. 20) (14.9 μmol, 81.7 mg), O-βAla-bohemine (14.9 μmol, 7.6mg), PyBOP (14.9 μmol, 7.8 mg), HOBt (14.9 μmol, 2.4 mg) and DIEA(0.2295 mmol, 29.7 mg) in DMF (2 mL) was stirred for 2 h. The peptidylresin was filtered, washed with DMF, CH₂Cl₂ and Et₂O and was dried invacuo (82 mg). The product was treated with cleavage reagent (5 mL, 2h). Crude product (42 mg) was obtained by precipitation with Et₂O andcentrifugation/decantation. It was purified by preparative RP-HPLC(0-60% MeCN gradient) to afford the pure title compound as a colourlesssolid (14.3 mg). Anal. RP-HPLC: t_(R)=14.8 min (0-60% MeCN gradient,purity>93%). DE MALDI-TOF MS: [M+H]⁺=2812.7 (C₁₃₂H₂₀₄N₄₂O₂₅S=2811.37).

Example 23 (Maleimidopropionoyl)bohemine

3-Maleimidopropionic acid (12.8 mg, 76 μmol) was dissolved in CH₂Cl₂ (1mL). The mixture was stirred and DIC (5.3 mg, 42 μmol) in dry CH₂Cl₂(0.5 mL) was added. The reaction was allowed to proceed with stirringfor 40 min. Solvent was then removed under reduced pressure. The residueof 3-maleimidopropionic acid anhydride was redissolved in dry pyridine(0.5 mL). A solution of bohemine (10.3 mg, 30 μmol) and DMAP (0.35 mg, 2μmol) in pyridine (0.5 mL) was added and the mixture was stirred underN₂ for 1 h. It was then evaporated to dryness under reduced pressure.The residue was redissolved in DMF (1 mL) and purified by preparativeRP-HPLC column (10-60% MeCN gradient) to afford the pure title compoundas a colourless solid (14.7 mg, 88%). Anal. RP-HPLC: t_(R)=17.7 min(0-60% MeCN gradient, purity>95%). ¹H-NMR (CDCl₃) and DE MALDI-TOF MSspectra were consistent with the proposed structure (C₂₅H₂₉N₇O₄=491.54).

O-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]bohemine(SEQ ID No. 27)

(Maleimidopropionoyl)bohemine (0.74 mg, 1.5 μmol) was dissolved in DMF(0.3 mL) and Et₃N (50 μL) was added.H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (3.5 mg, 1.5 μmol), dissolved in DMF (0.25 mL) was thenadded. The mixture was stirred under N₂ and was monitored by anal.RP-HPLC. After 1 h, the reaction was complete. The mixture was filteredand purified by preparative RP-HPLC (10-60% MeCN gradient) to afford thepure title compound as a colourless solid (1.7 mg, 40%). Anal. RP-HPLC:t_(R)=15.0 min (0-60% MeCN gradient; purity>95%). DE MALDI-TOF MS:[M+H]⁺=2842 (C₁₃₂H₂₀₂N₄₂O₂₅S₂=2841.42).

Example 24 4-(Iodoacetyl)podophyllotoxin

A mixture of podophyllotoxin (0.49 mmol, 204 mg), iodoacetic acid (1.03mmol, 192 mg), DIC (0.552 mmol, 69.7 mg) and DMAP (0.164 mmol, 20 mg) indry CH₂Cl₂ (5 mL) was cooled to 0° C. Pyridine (0.2 mL) was added andthe reaction mixture was allowed to stir for 1 h at 0° C. The mixturewas evaporated to dryness. The resulting light-yellow residue wasredissolved in MeCN and was purified by preparative RP-HPLC (20-70% MeCNgradient) to afford the pure title compound as a colourless solid (89.5mg). Anal. RP-HPLC: t_(R)=22.3 min (0-60% MeCN gradient, purity>95%).¹H-NMR (300 MHz, CDCl₃) δ: 2.85 (m, 2H, H2,3), 3.70 (s, 6H, OCH₃x2),3.72 (s, 2H, CH₂I), 3.74 (s, 3H, OCH₃), 4.13 (m, 1H, H11), 4.34 (m, 1H,H11), 4.53 (d, 1H, J=3.60 Hz, H1), 5.83 (d, 1H, J=8.43 Hz, H4), 5.93(dd, 2H, J=4.35, 1.17 Hz, OCH₂O)), 6.31 (s, 2H, H2′6′), 6.48 (s, 1H,H8), 6.77 (s, 1H, H5).

4-[Acetyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]podophyllotoxin(SEQ ID No. 27)

To a solution of 4-iodoacetyl)podophyllotoxin (17 μmol, 10 mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (6 μmol, 14 mg) in DMF (1 mL) was added Et₃N (0.9 μL, 6μmol). The mixture was stirred for 1 h. MeCN (0.5 mL) was added and thesolution was purified by preparative RP-HPLC (10-60% MeCN gradient) toafford the pure title compound as a colourless solid (9.9 mg, 59%).Anal. RP-HPLC: t_(R)=15.4 min (0-60% MeCN gradient, purity>97%). DEMALDI-TOF MS: [M+H]⁺=2806.8 (C₁₃₁H₁₉₅N₃₅O₃₀S₂=2804.30).

Example 254-[Acetyl-(H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]podophyllotoxin(SEQ ID No. 19)

A solution of 4-(iodoacetyl)podophyllotoxin (17 μmol, 10 mg) andH-Cys-bAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ (SEQ ID No. 19) (23 μmol,28.6 mg) in DMF (1 mL) was added Et₃N (2.4 μL, 17 μmol). After stirringfor 1 h MeCN (0.5 mL) was added and the mixture was purified bypreparative RP-HPLC (0-60% MeCN gradient) to afford the pure titlecompound as a colourless solid (29.4 mg, 100%). Anal. RP-HPLC:t_(R)=14.1 min (0-60% MeCN gradient, purity>98%). DE MALDI-TOF MS:[M+H]⁺=1661.0 (C₇₆H₁₁₄N₂₀O₁₈S₂=1659.97).

Example 26 4′-Demethyl-4-(iodoacetyl)epipodophyllotoxin

To a solution of 4′-demethylepipodophyllotoxin (0.26 mmol, 104 mg),iodoacetic acid (0.53 mmol, 98.8 mg), and DIC (0.32 mmol, 40.1 mg) inCH₂Cl₂ (2 mL) at 0° C. was added pyridine (50 μL) and DMAP (0.1 mmol,12.8 mg). After 1 h stirring the solvents were evaporated. The residuewas redissolved in DMF (1 mL) and purified by preparative RP-HPLC(20-60% MeCN gradient) to afford the pure title compound as a colourlesssolid (35.7 mg, 24%). Anal. RP-HPLC: t_(R)=20.3 min (0-60% MeCNgradient, purity>96%). ¹H-NMR (300 MHz, CDCl₃) δ: 3.02 (m, 1H, H3), 3.20(m, 1H, H2), 3.71 (s, 6H, OCH₃x2), 3.63 (s, 2H, CH₂I), 3.74 (s, 3H,OCH₃), 4.05 (m, 1H, H11), 4.27 (m, 1H, H11), 4.60 (d, 1H, J=4.94 Hz,H1), 6.06 (d, 1H, J=3.41 Hz, H4), 5.92 (m, 2H, OCH₂O), 6.21 (s, 2H,H2′6′), 6.49 (s, 1H, H8), 6.80 (s, 1H, H5).

4′-Demethyl-4-[acetyl-(H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]epipodophyllotoxin(SEQ ID No. 19)

To a solution of 4′-demethyl-4-(iodoacetyl)epipodophyllotoxin (17.6μvvmol, 10 mg) and H-Cys-βvAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ (SEQ IDNo. 19) (14.9 μmol, 18 mg) in DMF (1 mL) was added Et₃N (2.1 mL, 15μmol). After stirring for 1 h the reaction mixture was purified bypreparative RP-HPLC (0-60% MeCN gradient) to afford the pure titlecompound as a colourless solid (11.2 mg, 46%). Anal. RP-HPLC: t_(R)=12.8min (0-60% MeCN gradient, purity>98%). DE MALDI-TOF MS: [M+H]⁺=1647.2(C₇₅H₁₁₂N₂₀O₁₈S₂=1645.95).

Example 274′-Demethyl-4-[acetyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]epipodophyllotoxin(SEQ ID No. 27)

To a solution of 4′-demethyl-4(iodoacetyl)epipodophyllotoxin (22 μmol,12.6 mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂(SEQ ID No 27) (8 μmol, 20 mg) in DMF (1 mL) was added Et₃N (1.2 μL, 9μmol). After stirring for 1 h the mixture was purified by preparativeRP-HPLC (0-60% MeCN gradient) to afford the pure title compound as acolourless solid (13.3 mg, 56%). Anal. RP-HPLC: t_(R)=14.5 min (0-60%MeCN gradient, purity>96%). DE MALDI-TOF MS: [M+H]⁺=2789.5(C₁₃₀H₁₉₄N₃₆O₂₉S₂=2789.29).

Example 28 4-(Boc-Gly)podophyllotoxin

A mixture of podophyllotoxin (400 mg, 0.97 mmol), Boc-Gly-OH (510 mg,2.91 mmol) DIC (1.73 mmol, 273 μL), DMAP (0.41 mmol, 50 mg) and pyridine(173 μL) in CH₂Cl₂ (5 mL) was stirred at for 1 h. The solvents wereevaporated. The residue was redissolved in DMF (1.5 mL) and purified byRP-HPLC (20-70% MeCN gradient) to afford the pure title compound as acolourless solid (502.6 mg, 91%). Anal. RP-HPLC: t_(R)=22.1 min (0-60%MeCN gradient, purity>97%).

4-(H-Gly)podophyllotoxin

To a solution of 4-(Boc-Gly)podophyllotoxin (0.24 mmol, 137 mg) inCH₂Cl₂ (8 mL) was added TFA (0.5 mL). After stirring for 1 h thesolvents were evaporated. The resulting light-yellow solid residue waspurified by preparative RP-HPLC (10-70% MeCN gradient) to afford thepure title compound as a colourless solid (41.7 mg, 37%). Anal. RP-HPLC:t_(R)=15.2 min (0-60% MeCN gradient, purity>97%).

4-Maleimidopropionoyl-Gly)podophyllotoxin

To a solution of 3-maleimidopropionic acid (70 μmol, 11.8 mg) and DIC(38 μmol, 4.83 mg) in DMF (1 mL) was added 4-(H-Gly)podophyllotoxin (17μmol, 8 mg), DMAP (10 μmol, 1.2 mg) and pyridine (20 μL). After stirringfor 1 h the mixture was purified by preparative RP-HPLC (0-60% MeCNgradient) to afford the pure title compound as a colourless solid (1.1mg). Anal. RP-HPLC: t_(R)=18.2 min (0-60% MeCN gradient, purity>97%).

4-[(Succinimidopropionoyl-Gly)-(H-Cys-bAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]podophyllotoxin(SEQ ID No. 19)

To a solution of 4-(maleimidopropionoyl-Gly)podophyllotoxin (1.8 μmol,1.1 mg) and H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ (SEQ ID No. 19)(4 μmol, 5 mg) in DMF (1 mL) was added Et₃N (0.5 μL, 4 μmol). Themixture was stirred for 1 h. It was diluted with MeCN (0.5 mL) andpurified by preparative RP-HPLC (0-60% MeCN gradient) to afford thetitle compound as a colourless solid (1.1 mg, 33%). Anal. RP-HPLC:t_(R)=14.7 min (0-60% MeCN gradient, purity>97%). DE MALDI-TOF MS:[M+H]=1829.8 (C₈₃H₁₂₂N₂₂O₂₁S₂=1828.12).

Example 29 10-O-(Maleimidopropionoyl)camptothecin

To a solution of 10-hydroxycamptothecin (40 μmol, 14.7 mg),3-maleimidopropionic acid (0.228 mmol, 38.5 mg) and DIC (0.125 mmol,15.8 mg) in CH₂Cl₂ (2 mL) was added pyridine (0.2 mL). After stirringfor 1 h, the mixture was evaporated to dryness. The resultinglight-yellow solid was redissolved in DMF (1 mL) and purified bypreparative RP-HPLC (10-70% MeCN gradient) to afford the pure titlecompound (9.2 mg, 45%) as a light-yellow solid. Anal. RP-HPLC:t_(R)=15.7 min (0-60% MeCN gradient, purity>97%). ¹H-NMR (300 MHz,CDCl₃) δ: 1.05 (t, 3H, J=7.5 Hz, CH₃), 1.91 (m, 2H, J=7.8 Hz, CH₂), 2.98(t, 2H, J=7.8 Hz, CH₂), 4.04 (t, 2H, J=7.8 Hz, CH₂), 5.32 (m, 3H, H5,H17), 6.77 (s, 2H, CH═CH), 7.60 (m, 1H, H11), 7.72 (m, 2H, H14, H9),8.24 (d, 1H, J=9.2 Hz, H12), 8.36 (s, 1H, H7).

10-O-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]camptothecin(SEQ ID No. 27)

To a solution of 10-O-(maleimidopropionoyl)camptothecin (9 μmol, 4.6 mg)andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂(SEQ ID No. 27) (4 μmol, 10 mg) in DMF (1 mL) was added Et₃N (0.55 μL, 4μmol). After stirring for 1 h, the mixture was purified by preparativeRP-HPLC (0-60% MeCN gradient) to afford the pure title compound as acolourless solid (6.5 mg, 57%). Anal. RP-HPLC: t_(R)=14.0 min (0-60%MeCN gradient, purity>98%). DE MALDI-TOF MS: [M+H]⁺=2864.7(C₁₃₄H₁₉₅N₃₉O₂₈S₂=2864.36).

Example 30 H-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH₂ (SEQ ID No. 21)

Starting from Rink Amide AM resin (0.69 mmol/g, Novabiochem),H-Cys(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-Cys(Trt)-resin(SEQ ID No. 21) was assembled. After deprotection (1.5 h), the crudepeptide was obtained by precipitation from Et₂O,centrifugation/decantation, and drying. Aliquots (total 258 mg) werepurified by preparative RP-HPLC (9-19% MeCN gradient) to afford the puretitle compound (132.4 mg). Anal. RP-HPLC: t_(R)=20.3 min (8-18% MeCNgradient, purity>99%, λ=214 nm). DE MALDI-TOF MS: [M+H]⁺=1238.6(C₅₂H₉₂N₂₀O₉S₃=1237.63).

Bis-[4-(succinimidopropionoyl)podophyllotoxin]-(H-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH₂)(SEQ ID No. 21)

To a solution of 4-(maleimidopropionoyl)podophyllotoxin (19 μmol, 11 mg)and H-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH₂ (SEQ ID No. 21) (12 μmol,15 mg), in DMF (1 mL) was added Et₃N (2.8 μL). After stirring for 1 hthe mixture was purified by preparative RP-HPLC (10-70% MeCN gradient)to afford the pure title compound as a colourless solid (9.0 mg, 32%).Anal. RP-HPLC: t_(R)=17.4 min (0-60% MeCN gradient, purity>98%). DEMALDI-TOF MS: [M+H]⁺=2369.7 (C₁₁₀H₁₄₆N₂₂O₃₁S₃=2368.66).

Example 314′-(Succinimidopropionoyl)epipodophyllotoxin-(H-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH₂)-10-O-(succinimidopropionoyl)camptothecin(SEQ ID No. 21)

To a solution of 10-O-(maleimidopropionoyl)camptothecin (0.005 mmol, 2.6mg), 4′-(maleimidopropionoyl)epipodophyllotoxin (5.6 μmol, 3.1 mg), andH-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH₂ (SEQ ID No. 21) (11 μmol, 13mg), in DMF (1.5 mL) was added Et₃N (1.5 μL). After stirring for 1.5 hthe mixture was purified by preparative RP-HPLC (10-70% MeCN gradient)to a afford the pure title compound as a colourless solid (1.9 mg).Anal. RP-HPLC: t_(R)=14.8 min (0-60% MeCN gradient, purity>96%). DEMALDI-TOF MS: [M+H]⁺=2304.6 (C₁₀₇H₁₃₈N₂₄O₂₈S₃=2304.58).

Example 324′-(Succinimidopropionoyl)epipodophyllotoxin-(H-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH₂)-2′-(succinimidopropionyl)paclitaxel(SEQ ID No. 21)

To a solution of4′-[succinimidopropionoyl-(H-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH₂)]epipodo-phyllotoxin(SEQ ID No. 21) (2 μmol, 3.5 mg), 2′-(maleimidopropionyl)paclitaxel (2μmol, 2 mg) in DMF (1 mL) was added Et₃N (0.3 μL). After stirring for1.5 h the reaction mixture was purified by preparative RP-HPLC (10-70%MeCN gradient) to afford the pure title compound as a colourless solid(1.5 mg). Anal. RP-HPLC: t_(R)=17.8 min (0-60% MeCN gradient,purity>98%). DE MALDI-TOF MS: [M+H]⁺=2794.5 (C₁₃₄H₁₇₃N₂₃O₃₇S₃=2794.14).

Example 33 4′-Methoxy-(4″-aminoanilino)epipodophyllotoxin and4′-demethyl-(4″-aminoanilino)epipodophyllotoxin

A solution of podophyllotoxin (3.6 mmol, 1.5 g) in ClCH₂CH₂Cl (15 mL)was kept at 0° C. and HBr gas was passed through the solution. After 45min N₂ was passed through the reaction mixture to drive off excess HBr.To this solution anhydrous barium carbonate (4.32 mmol, 0.85 g) and4-nitroaniline (4.32 mmol, 0.6 g) were added. The mixture was stirred atambient temperature for 18 h under N₂. It was diluted with EtOAc andfiltered. The filtrate was washed with water, dried on MgSO₄, andpurified by flash, chromatograph (100:5:5 CH₂Cl₂/EtOAc/acetone) toafford crude 4′-methoxy-(4″-nitroanilino)epipodophyllotoxin and4′-demethyl-(4″-nitroanilino)epipodophyllotoxin. Further purification bypreparative RP-HPLC (10-70% MeCN gradient) afforded the pure products asyellow solids.

4′-Methoxy-4-(4″-nitroanilino)epipodophyllotoxin

Anal. RP-HPLC: t_(R)=22.3 min (0-60% MeCN gradient, purity>95%). ¹H-NMR(300 MHz, CDCl₃) δ: 3.09 (m, 2H, H2,3), 3.77 (s, 6H, OCH₃), 3.83 (s, 3H,OCH₃), 3.86 (m, 1H, H11), 4.42 (m, 1H, H11), 4.63 (m, 2H, H1,4), 4.84(m, 1H, NH), 6.00 (m, 2H, OCH₂O), 6.31 (s, 2H, H2′,6′), 6.57 (m, 3H, H8,Ar), 6.76 (s, 1H, H5), 8.16 (d, 2H, J=9.08 Hz, Ar).

4′-Demethyl-4-(4″-nitroanilino)epipodophyllotoxin

Anal. RP-HPLC for: t_(R)=20.5 min (0-60% MeCN gradient, purity>95%).¹H-NMR (300 MHz, CDCl₃) δ: 3.07 (m, 2H, H2,3), 3.79 (s, 6H, OCH₃), 3.81(m, 1H, H11), 4.40 (m, 1H, H11), 4.60 (m, 2H, H1), 4.73 (m, 1H, H4),4.83 (m, 1H, NH), 5.45 (br, 1H, OH), 5.98 (m, 2H, OCH₂O), 6.31 (s, 2H,H2′,6′), 6.57 (m, 3H, H8, Ar), 6.76 (s, 1H, H5), 8.14 (d, 2H, J=9.04 Hz,Ar).

To a solution of 4′-methoxy-4-(4″-nitroanilino)epipodophyllotoxin or4′-demethyl-(4″-nitroanilino)epipodophyllotoxin in 10:1 EtOAc/MeOH wasadded 10% palladium on activated carbon. The mixture was stirred underH₂ for 3 h. The catalyst was filtered and washed several times withMeOH. The combined filtrate and washing were evaporated to dryness togive a light-yellow solid which was redissolved in MeCN and purified bypreparative RP-HPLC (10-70% MeCN gradient) to afford the products asyellow solids in a quantitative yield.

4′-Methoxy-4-(4″-aminoanilino)epipodophyllotoxin

Anal. RP-HPLC: t_(R)=16.1 min (0-60% MeCN gradient, purity>95%). ¹H-NMR(300 MHz, CDCl₃) δ: 287 (m, 1H, H3), 3.11 9m, 1H, H2), 3.68 (s, 6H,OCH₃), 3.73 (s, 3H, OCH₃), 3.61 (m, 1H, H11), 4.15 (m, 1H, H11),4.52-4.62 (m, 2H, H1,4), 5.86 (m, 2H, OCH₂O), 6.28 (s, 2H, H2′,6′), 6.37(m, 2H, Ar), 6.45 (s, 1H, H8), 6.69 (s, 1H, H5), 7.03 (m, 2H, Ar).

4′-Demethyl-4-(4″-aminoanilino)epipodophyllotoxin

Anal. RP-HPLC: t_(R)=14.2 min (0-60% MeCN gradient, purity>97%). ¹H-NMR(300 MHz, CDCl₃) δ: 3.05 (m, 1H, H3), 3.18 (m, 1H, H2), 3.78 (s, 6H,OCH₃), 3.93 (m, 1H, H11), 4.38 (m, 1H, H11), 4.60 (d, 1H, J=5.91 Hz,H1), 4.70 (d, 1H, J=3.86 Hz, H4), 5.96 (m, 2H, OCH₂O), 6.33 (s, 2H,H2′6′), 6.53 (s, 1H, H8), 6.62 (d, 2H, J=8.66 Hz, Ar), 6.75 (s, 1H, H5),7.19 (d, 2H, J=8.60 Hz, Ar).

4′-Methoxy-4-[4″-aminoanilino-maleimidopropionoyl)]epipodophyllotoxin

To a solution of 4′-methoxy-4-(4″-aminoanilino)epipodophyllotoxin (41μmol, 20.8 mg), 3-maleimidopropionic acid (0.226 mmol, 38.2 mg), DIC(0.124 mmol, 15.7 mg) and DMAP (40 μmol, 4.9 mg) in CH₂Cl₂ (2 mL) wasadded pyridine (0.2 mL). After stirring for 1 h, the mixture wasevaporated to dryness. The resulting light-yellow solid was redissolvedin DMF (1 mL) and purified by preparative RP-HPLC (20-70% MeCN gradient)to afford the pure title compound as a colourless solid (10.1 mg, 38%).Anal. RP-HPLC: t_(R)=19.5 min (0-60% MeCN gradient, purity>96%). ¹H-NMR(300 MHz, CDCl₃) δ: 2.71 (t, 2H, J=7.0 Hz, CH₂), 2.91 (m, 1H, H3), 3.14(m, 1H, H2), 3.76 (s, 6H, OCH₃), 3.82 (s, 3H, OCH₃), 3.93 (t, 2H, J=7.0Hz, CH₂), 3.97 (m, 1H, H5, H11), 3.49 (m, 1H, H11, 4.63 (m, 2H, H1,4),5.97 (m, 2H, OCH₂O), 6.32 (s, 2H, H2′6′), 6.50 (m, 2H, Ar), 6.53 (s, 1H,H8), 6.73 (s, 2H, CH═CH), 6.74 (s, 1H, H5), 7.32 (m, 2H, Ar).

4′-Methoxy-4-[4″-aminoanilino-(succinimidopropionoyl)-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]epipodophyllotoxin(SEQ ID No. 27)

To a solution of4′-methoxy-4-[4″-aminoanilino-(maleimidopropionoyl)]epipodophyllotoxin,(6 μmol, 4.1 mg) andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂(SEQ ID No. 27) (6 μmol, 14 mg) in DMF (1 mL) was added Et₃N (2 μL).After stirring for 1 h, the mixture was purified by preparative RP-HPLC(0-60% MeCN gradient) to afford the pure title compound as a colourlesssolid (5.8 mg, 32%). Anal. RP-HPLC: t_(R)=16.0 min (0-60% MeCN,purity>99%). DE MALDI-TOF MS: [M+H]⁺=3003.9 (C₁₄₂H₂₀₇N₃₉O₃₀S₂=3004.54).

Example 344′-Methoxy-4-[4″-aminoanilino-(succinimidopropionoyl)-(H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]epipodophyllotoxin(SEQ ID No. 19)

To a solution of4′-methoxy-[4″-aminoanilino-(maleimidopropionoyl)]epipodophyllotoxin (7μmol, 4.6 mg) and H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ (SEQ ID No.19) (14 μmol, 16.3 mg) in DMF (1 μL) was added Et₃N (1 mL). Afterstirring for 1 h, the mixture was purified by preparative RP-HPLC (0-60%MeCN gradient) to afford the pure title compound as a colourless solid(6.4 mg, 49%). Anal. RP-HPLC: t_(R)=15.2 min (0-60% MeCN gradient,purity>98%). DE MALDI-TOF MS: [M+H⁺=1861.6 (C₈₇H₁₂₅N₂₃O₁₉S₂=1861.20).

Example 354′-Demethyl-4-[4″-aminoanilino-(maleimidopropionoyl)]epipodophyllotoxin

To a solution of 4′-demethyl-4-(4″-aminoanilino)epipodophyllotoxin (24μmol, 12 mg), 3-maleimidopropionic acid (49 μmol, 8.3 mg), and DIC (27μmol, 3.4 mg) in 1:1 DMF/CH₂Cl₂ (2 mL) was added pyridine (10 μL). Afterstirring for 1 h, the reaction mixture was evaporated. The resultinglight-yellow solid was purified by preparative RP-HPLC (10-70% MeCNgradient) to afford the pure title compound as a colourless solid (5.3mg, 34%). Anal. RP-HPLC: t_(R)=19.5 min (0-60% MeCN gradient,purity>96%). ¹H-NMR (300 MHz, CDCl₃) δ: 2.65 (t, 2H, J=7.3 Hz, CH₂),2.98 (m, 1H, H3), 3.17 (m, 1H, H2), 3.79 (s, 6H, OCH₃), 3.93 (t, 2H,J=7.0 Hz, CH₂), 3.99 (m, 1H, H5, H11), 4.38 (m, 1H, H11), 4.58 (d, 1H,J=4.95 Hz, H1), 4.64 (d, 1H, J=3.95 Hz, H4) 5.96 (m, 2H, OCH₂O), 6.33(s, 2H, H2′6′), 6.49-6.53 (m, 3H, H8, Ar), 6.74 (s, 2H, CH═CH), 6.75 (s,1H, H5), 7.33 (m, 2H, Ar).

4′-Demethyl-4-[4″-aminoanilino-(succinimidopropionoyl)-(H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]epipodophyllotoxin(SEQ ID No. 19)

To a solution of4′-demethyl-[4″-aminoanilino-(maleimidopropionoyl)]epipodophyllotoxin(8.3 μmol, 5.3 mg) and H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH2 (SEQID No. 19) (13 μmol, 15.6 mg) in DMF (1.5 mL) was added Et₃N (2 μL).After stirring for 1 h, the mixture was purified by preparative RP-HPLC(0-60% MeCN gradient) to afford the pure title compound as a colourlesssolid (14.9 mg, 97%). Anal. RP-HPLC: t_(R)=13.7 min (0-60% MeCNgradient, purity>98%). DE MALDI-TOF MS: [M+H]⁺=1847.1(C₈₆H₁₂₃N₂₃O₁₉S₂=1847.17).

Example 36 In vitro cytotoxic activity of{[4[N-(2,4-diamino-6-pteridinyl-methyl)-N-methylamino]benzoyl]-Glu-Gly-βAla}₄-Lys₂-Lys-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 28)

This compound (abbreviated ‘MTX-Pen’ in the tables below) was evaluatedfor its ability to inhibit cell proliferation of normal (immortalised)human cells (HaCaT cells, Tables 1 & 2) and a human colorectal cancercell line (HT29, Table 3). The free drug methotrexate (‘MTX’ in Table1-3) and the free vectorH-Ala-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(abbreviated ‘Pen’ in Table 1 below) were included for the purposes ofcomparison.

Assay procedure—Cells were seeded into 96-well plates at 2,500cells/well in DMEM with 10% FCS and antibiotics. After overnightincubation, test compound dilutions in cell medium were prepared andwere added to the cells. Samples were taken 1, 2, 3, and 4 days aftercompound addition. Nucleotide Releasing Reagent (LumiTech ViaLight kit)was added in order to lyse the cells and release ATP. After incubationat room temperature (5 min), the mixtures were transferred to opaque96-well plates and stored at −20° C. until analysis. After loadingplates into a luminometer (Lucy 1, Labtech International), ATPMonitoring Reagent (20 μL/well, LumiTech ViaLight kit) was added to eachwell successively and light intensity was measured immediately. Sixreadings were taken per sample. Each assay point was established usingsix replicates and appropriate controls. ATP bioluminescence was foundto be proportional to viable cell count over the entire cells/well rangeused. Statistically significant results in the tables below are printedin bold face.

TABLE 1 (HaCaT Cells) % Cell Death Day 1 Day 2 Day 3 Day 4 Dose (μM) MTXMTX-Pen Pen MTX MTX-Pen Pen MTX MTX-Pen Pen MTX MTX-Pen Pen 40.0 4 29 1615 82 −22 79 97 5 92 98 12 13.3 22 −42 18 35 63 0 82 97 −17 92 98 −6 4.44 −8 8 24 45 −4 77 95 −1 93 98 10 1.5 13 −24 16 31 82 −31 77 82 2 94 88−14 0.5 −4 −19 6 31 2 −6 75 29 −29 93 49 −26 0.2 7 14 26 11 21 0 79 20−3 93 51 21

TABLE 2 (HaCaT Cells) % Cell Death Day 1 Day 2 Day 3 Day 4 Dose MTX-MTX- MTX- MTX- (μM) MTX Pen MTX Pen MTX Pen MTX Pen 40.0 42 88 95 9413.3 27 87 95 94 4.4 21 15 70 52 97 95 92 88 1.5 14 19 67 12 96 −16 9117 0.5 0 13 59 24 96 −27 91 2 0.2 3 41 94 86 0.1 19 7 45 65

TABLE 3 (HT 29 Cells) % Cell Death Day 1 Day 2 Day 3 Day 4 Dose MTX-MTX- MTX- MTX- (μM) MTX Pen MTX Pen MTX Pen MTX Pen 40.0 31 79 96 9813.3 3 45 88 96 4.4 −14 10 −4 6 58 46 86 77 1.5 17 16 −5 9 48 15 84 450.5 15 14 −12 8 52 17 88 16 0.2 10 −5 54 85 0.1 6 −17 52 84

Example 37 Stabilisation of microtubule formation by paclitaxel and2′-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]paclitaxel(SEQ ID No. 27)

Assay procedure.—A solution of bovine tubulin andtetramethylrhodamine-labeled tubulin (total concentration 0.5 mg/mL) inG-PEM buffer (80 mM PIPES, pH 6.8, 1 mM EDTA, 1 mM GTP) was incubated inthe presence of 10 μM paclitaxel, 10 μM2′-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]paclitaxel(SEQ ID No. 27), or without test compound for 30 min at 37° C. Formationof microtubules was visualised on a Nikon Eclipse E800 fluorescencemicroscope. Images were captured with a Kodak DCS 420 digital camera andanalysed using Adobe 5.0 software. Microtuble formation was stabilizedusing the delivery systems of the present invention.

Example 38 Internalisation of4-[succinimidopropionoyl-(biotinamidocaproyl-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂)]podophyllotoxin(SEQ ID No. 18) into cells

A549 cells were seeded into 96-well plates at 50,000 cells per well inDMEM with 10% FCS and antibiotics. After overnight incubation,4-[succinimidopropionoyl-(biotinamidocaproyl-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂)]podophyllotoxin(SEQ ID No. 18) (labelled ‘conjugate’ in FIG. 1 below) orbiotinamidocaproyl-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂(SEQ ID No. 23) (labelled ‘vector’) were prepared as a dilution seriesof six decreasing concentrations in cell medium and were added to cells.At the end of the incubation period (60 min), the cells were rinsedthree times with PBS and fixed for 20 min at −20° C. in ethanol/aceticacid (95/5). After the fixation, the cell were permeabilised for 10 minwith PBS containing 3% Tween-20. Endogenous alkaline phosphatase wasneutralised by incubating the plate at 65° C. for 60 min. Cells wereincubated for 30 min at room temperature with alkalinephosphatase-streptavidine (Pierce Chemical Co.) in 0.1% BSA in PBS andwere washed extensively with PBS. A freshly made solution of 1 mg/mLnitrophenyl phosphate in 10 mM diethanolamine (pH 9.5), 0.5 mM MgCl₂ wasadded to each well and incubated until sufficient colour developed(approximately 30 min). The enzymatic reaction was stopped by adding 50μl 2 M aq NaOH. Alkaline phosphatase activity was measuredspectrophotometrically at 405 nm.

Example 39 Visualisation of cell internalisation by4-[Succinimidopropionoyl-(biotinamidocaproyl-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂)]podophyllotoxin(SEQ ID No. 18)

Cells were seeded into 8-well chamber slides at 50,000 cells per well inDMEM with 10% foetal calf serum and antibiotics. After overnightincubation,4-[(Succinimidopropionoyl-(biotinamidocaproyl-βAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂)]podophyllotoxin(SEQ ID No. 18) was prepared in cell medium at a concentration of 10 μMand was added to cells. At the end of the incubation period (60 min),the cells were rinsed three times with PBS and fixed for 20 min at −20°C. in ethanol/acetic acid (95/5). After the fixation, the cells werepermeabilised for 10 min with PBS containing 3% Tween-20. The slideswere incubated with streptavidin-FITC (Pierce Chemical Co.), diluted inPBS for 30 min. at room temperature, washed extensively with PBS andmounted in Hydromount (BDH). The distribution of the fluorescence wasanalysed on a Nikon Eclipse E800 fluorescence microscope. Images werecaptured with a Kodak DCS 420 digital camera and analysed using Adobe5.0 software. Using these techniques, the internalization of a deliverysystem of the present invention can be monitored.

Example 40 Intracellular stability of4-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]podophyllotoxin(SEQ ID No. 27)

10×10⁶ HL60 cells were incubated for 1 h with 15 μM4-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]podophyllotoxin(SEQ ID No. 27) (A in FIG. 2 below) or without test compound (B) inDMEM. After the incubation, cells were washed extensively with PBS untilno test compound was detectable in the washes. Cells were incubated forone additional h with neat medium. Afterwards the cells were pelleted,resuspended in 50 mM Tris pH 7.5, containing a cocktail of proteaseinhibitors and they were solubilised by ultrasonication for 1 min. Theinsoluble fraction was pelleted for 15 min using an Eppendorf centrifugeand the supernatant was analysed by anal. RP-HPLC (0-60% MeCN gradient,λ=254 nm). Intact test compound was identified by reference tochromatograms obtained with authentic test compound and by DE MALDI-TOFMS analysis of the peak fraction indicated with an arrow in FIG. 2.Pellets were further extracted with DMSO and extracts analysedsimilarly, no test compound was detected.

Example 41 Serum Stability of Peptide Vectors

Test compounds were dissolved in cell-conditioned tissue culture medium(10% FCS in DMEM) at concentrations varying from 1 to 40 μM. Thesolutions were incubated at 37° C. and samples were withdrawn atintervals. After filtration, aliquots were analysed by RP-HPLC (using aphotodiode array UV detector). Intact vectors were identified byreference to chromatograms obtained with authentic peptides and by DEMALDI-TOF analysis of appropriate peak fractions. The half-lives forfour different vectors are summarised in Table 4. Similar results wereobtained when human or murine serum was substituted for bovine serum(FCS). The latter was chosen preferentially in order to replicate theconditions used for cytotoxicity assays on cell cultures. In all casesthe main metabolism product of the 16mer peptide acidH-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 25) was the 15mer resulting from C-terminal truncation of aLys residue. This 15mer peptide was observed to survive for several hprior to further carboxy-terminal degradation. The L-aminoacid-containing vector peptide amides were degraded much more slowly, noindividual metabolites could be identified. All D-amino acid-containingpeptide vectors studied were very stable and could usually still bedetected after 72 h incubations.

TABLE 4 Serum Vector t_(1/2) H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-10 min Arg-Met-Lys-Trp-Lys-Lys-OHH-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg- >12 hArg-Met-Lys-Trp-Lys-Lys-NH₂H-D-Arg-D-Gln-D-Ile-D-Lys-D-Ile-D-Trp-D-Phe- >24 hD-Gln-D-Asn-D-Arg-D-Arg-D-Met-D-Lys-D-Trp-D- Lys-D-Lys-NH₃H-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ 3 h(Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lyscorresponds with SEQ ID No. 25 and Arg-Arg-Met-Lys-Trp-Lys-Lyscorresponds with SEQ ID No. 26)

Example 42 Serum Stability of Drug-ester Linkages

Test compounds (Table 5: A,4-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]podophyllotoxin(SEQ ID No. 27); B,4-[acetyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]podophyllotoxin(SEQ ID No. 27); C,2′-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]paclitaxel)(SEQ ID No. 27) were dissolved in cell-conditioned tissue culture medium(10% FCS in DMEM) at concentrations varying from 1 to 40 μM. Thesolutions were incubated at 37° C. and samples were withdrawn atintervals. After filtration, aliquots were analysed by RP-HPLC (using aphotodiode array UV detector). Hydrolysis of the ester bonds between thedrug hydroxy groups and the linker carboxyl groups was assessed byappearance of free podophyllotoxin or paclitaxel. The half-lives forthree different drug-linker combinations are summarised in Table 5.Similar results were obtained when human or murine serum was substitutedfor bovine serum (FCS). The latter was chosen preferentially in order toreplicate the conditions used for cytotoxicity assays on cell cultures.

TABLE 5 Serum t_(1/2) of Drug-Linker Entry Structure Ester bond A

>24 h B

40 min C

>12 h

Example 43 Comparison of Cytotoxic Activities of Paclitaxel and itsVector Conjugates

In order to demonstrate the cytotoxic biological effect on cancer cells(A549 lung carcinoma and MCF7 breast carcinoma cell lines in Table 6) ofthe paclitaxel-conjugates (paclitaxel-(16mer vector),2′-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH₂)]paclitaxel(SEQ ID No. 30); paclitaxel-(7mer vector),2′-[succinimidopropionoyl-(H-Cys-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]paclitaxel)(SEQ ID No. 19), cells were exposed to test compounds for 1 h only, i.e.a period during which the conjugates were shown to be metabolicallystable under the conditions of the assay procedures (refer Examples 41 &42). IC₅₀ values for 1-h and 3-d exposures are summarised in Table 6 andare compared with those obtained with free paclitaxel. It should benoted that due to the negligible water-solubility of unconjugatedpaclitaxel, washing off of compound not internalised into the cellsafter exposure was much less effective than for the conjugates, whichhave solubility in physiological media of >10 mg/mL. It can be concludedthat for the 1-h exposure results, the full cytotoxic activity can beattributed to the intact paclitaxel-conjugates (refer also Example 37).

Assay procedure—Cells were seeded into 96-well plates at 2,500 cells perwell in DMED, containing 10% FCS and antibiotics. After overnightincubation, test compounds were prepared as dilution series in cellmedium (addition of dimethylsulfoxide in the case of free paclitaxel toeffect partial dissolution) and were added to the cells. For the 1-hexposure samples, incubation was continued for 1 h, cell culture mediumsupernatants were removed and the wells were further washed with cellculture medium (5×2 min). Total viable cells were quantitated after atotal of 72 h incubation using a standard MTT-assay.

TABLE 6 72-h IC₅₀ (μM) Cell line A549 MCF7 Exposure time Test compound 1h 3 d 1 h 3 d Paclitaxel 0.028 <0.015 0.04  <0.015 Paclitaxel-(16 mervector) 0.618 <0.015 0.202   0.017 conjugate Paclitaxel-(7 mer vector)0.043 <0.015 0.325 <0.015 conjugate

Example 44 Evaluation of Paclitaxel- and Podophyllotoxin-vectorConjugates in Carcinoma Cell Line Panel

Serial dilutions of test compounds (Table 7: 2′-paclitaxel vectorconjugate,2′-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]paclitaxel(SEQ ID No. 27); 7-paclitaxel vector conjugate,7-[succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]paclitaxel(SEQ ID No. 27); 4-podophyllotoxin vector conjugate,4-[Succinimidopropionoyl-(H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)]podophyllotoxin)(SEQ ID No. 27) were applied to the cell lines. After incubation for 96h, cytotoxicity was assessed using a standard SRB-assay.

TABLE 7 96-h IC₅₀ (μM) cytotoxicity evaluation in cell line panel 2′- 4-Paclitaxel 7-Paclitaxel Podophyllotoxin Vector Vector Vector Cell LineVector Conjugate Conjugate Paclitaxel Conjugate Etoposide BE >25 0.03051.6 <0.0025 0.55 1.1 COLO205 >25 0.074 1.9 0.0026 0.495 0.8 DLD-1 >250.6 25 0.054 0.65 0.57 HCT116 >25 0.096 2.4 <0.0025 0.53 1.9 HT29 >250.092 2.25 <0.0025 0.53 2.6 KM12 >25 0.105 2.95 0.00285 0.58 0.58LIM1215 >25 0.12 3.65 0.0058 1.1 0.33 LS174T >25 0.195 7.4 0.0085 1.250.46 A2780 >25 0.105 2.8 <0.0025 0.54 0.21 A2780Cis^(R) >25 0.125 4.30.0051 0.54 0.68 CH1 >25 0.115 6.6 0.00415 0.51 0.165 CH1Dox^(R) >254.6 >25 0.54 0.51 6.6 CH1Taxol^(R) >25 0.13 8.7 0.0058 0.52 0.145SKOV-3 >25 0.235 22 0.01 0.74 13

Example 45 Evaluation of Etoposide and Podophyllotoxin Derivatives inTopoisomerase II Inhibition Assay

Topoisomerase II assay—Plasmid DNA (0.3 μg) was incubated at 37° C. with4 units of purified recombinant human topoisomerase II in cleavagebuffer (30 mM Tris.HCl, pH 7.6, 60 mM NaCl, 3 mM ATP, 15 mMmercaptoethanol, 8 mM MgCl₂) with or without the addition of testcompound (at 1 mM, 100 μM, or 10 μM final concentration). Reactions werestopped by the immediate addition of SDS (1% w/v final). Samples weretreated with proteinase K (30 min at 37° C.) and extracted twice with anequal volume of 42:1 CHCl₃/i-amyl alcohol. After adding loading dye,samples were loaded to a 4×TAE, 1% agarose gel containing 0.5 mg/mLethidium bromide and electrophoresed for 16-24 h. Topoisomerase IIinhibition was judged by the production of linear plasmid DNA,representing trapped cleavage intermediate, and by the ratio ofsubstrate (spercoiled DNA) to product (relaxed DNA). A relaxation assaywas performed identically, except that the reaction buffer was optimisedfor the detection of catalysis rather than cleavage, i.e. only 2 unitsof enzyme were used per sample. The reaction buffer was 50 mM Tris.HCl,pH 8, 120 mM KCl, 0.5 mM ATP, 0.5 mM dithiothreitol, 10 mM MgCl₂.Topoisomerase II inhibition was judged by the ratio of substrate(supercoiled DNA) to product (relaxed DNA).

TABLE 8 Activity Test Compound observed^(a) Etoposide IC Podophyllotoxin— 4′-Demethylepipodophyllotoxin IC4′-Demethyl-4-(4″-aminoanilino)epipodophyllotoxin IH-βAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂ —4-[Succinimidopropionoyl-(H-Cys-βAla-Arg-Arg-Met-Lys- —Trp-Lys-Lys-NH₂)]podophyllotoxin4′-[Succinimidopropionoyl-(H-Cys-βAla-Arg-Arg-Met-Lys- ICTrp-Lys-Lys-NH₂)]epipodophyllotoxin4′-Demethyl-4-[acetyl-(H-Cys-βAla-Arg-Arg-Met-Lys- ICTrp-Lys-Lys-NH₂)]epipodophyllotoxin4′-Demethyl-4-[4″-aminoanilino-(succinimidopropionoyl)- I(H-Cys-bAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH₂)]- epipodophyllotoxin ^(a)Idenotes inhibition of relaxation of supercoiled plasmid by topoisomeraseII. C denotes accumulation of topoisomerase II reaction intermediate.(Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys corresponds with SEQ ID No. 24 andCys-Ala-Arg-Meg-Lys-Trp-Lys-Lys corresponds with SEQ ID No. 19)

EXAMPLES

Abbreviations Boc tert-Butyloxycarbonyl Bu^(t) tert-Butyl CF₃COOHTrifluoroacetic acid CH₂Cl₂ Dichloromethane DE MALDI-TOF MS Delayedextraction matrix-assisted laser desorption ionisation time-of-flightmass spectrometry DMF N,N-Dimethylformamide Et₂O Diethyl ether Fmoc9-Fluorenylmethyloxycarbonyl HMBA p-Hydroxymethylbenzoyl HOBt1-Hydroxybenzotriazole MeCN Acetonitrile Pmc2,2,5,7,8-Pentamethylchroman-6-sulphonyl Pr^(i) ₂NEtN,N-Diisopropylethylamine PyBOP Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate RP-HPLC Reversed-phase high performanceliquid chromatography Trt Trityl (triphenylmethyl)

Example 1aH-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-Resin(SEQ 11 No. 25)

Peptide assembly was performed using an ABI 433A Peptide Synthesizer(Perkin-Elmer Applied Biosystems). A standard synthesis protocol(“FastMoc 0.25 mmol MonPrevPk”) was applied. The starting resin wasFmoc-Lys(Boc)-[(4-(hydroxymethyl)pheneoxyacetyl)-Resin] (ABI 401425; 0.5mmol/g). The final peptidyl resin (1.37 g; 100%) was washed with Et₂Oand dried in vacuo.

In order to demonstrate the chemical integrity of this intermediate, asmall aliquot of peptidyl resin was cleaved and deprotected, followed byanalysis of the crude productH-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 1), which revealed purity of >90% (anal. RP-HPLC) andchemical identity (DE MALDI-TOF MS and quantitative amino acidanalysis).

[H-Glu(OBu^(t))-Gly-bAla]₄-Lys₂-Lys-bAla-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-Resin(SEQ ID No. 28)

The above peptidyl resin (137 mg, 0.025 mmol) was acylated withFmoc-bAla-OH (47 mg, 0.15 mmol), PyBOP (78 mg, 0.15 mmol), HOBt (20 mg,0.15 mmol) and

Pr^(i) ₂NEt (39 mL, 0.225 mmol) in DMF (2 mL) during 2 h. It was thenFmoc-deprotected with 20% piperidine in DMF for 20 min and washedextensively with DMF. The product was further extended by two successiveacylation and deprotection cycles using Fmoc-Lys(Fmoc)-OH (0.15 mmol infirst cycle; 0.3 mmol in second cycle) using similar coupling anddeprotection steps. This was followed by further chain extension withFmoc-Gly-OH (0.6 mmol) and Fmoc-Glu(OBu^(t))-OH (0.6 mmol), again usingsimilar acylation and Fmoc-deprotection conditions. The product wasFmoc-deprotected and washed extensively with DMF, CH₂Cl₂ and Et₂O,followed by drying in vacuo.

In order to demonstrate chemical integrity of this intermediate, a smallaliquot of peptidyl resin was cleaved and side-chain deprotected,followed by analysis of the crude product[H-Glu-Gly-bAla]₄-Lys₂-Lys-bAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 28), which revealed purity (>89%; RP-HPLC on Vydac 218TP54,1 mL/min, 25° C., 15-25% MeCN in 0.1% aq CF₃COOH over 20 min, t_(R)=17.7min, l=200-300 nm) and identity (DE MALDI-TOF MS: [M+H]⁺=3732,C₁₆₅H₂₆₉N₅₃O₄₄S=3731.30).

{[4[N-(2,4-diamino-6-pteridinyl-methyl)-N-methylamino]benzoyl]-Glu(OBu^(t))-Gly-bAla}₄-Lys₂-Lys-bAla-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-Resin(SEQ ID No. 28)

The above peptidyl resin (76 mg, 0.025 mmol) was reacted overnight atroom temperature with4[N-(2,4-diamino-6-pteridinyl-methyl)-N-methylamino]benzoic acidhemihydrochloride dihydrate (Aldrich 86; 155-3; 76 mg, 0.2 mmol) andPyBOP (104 mg, 0.2 mmol), HOBt (27 mg, 0.2 mmol) and Pr^(i) ₂NEt (70 mL,0.4 mmol) in DMF (2 mL). The product was washed successively with DMF,CH₂Cl₂ and Et₂O and dried in vacuo to afford the title compound (85 mgorange peptidyl resin).

{[4[N-(2,4-diamino-6-pteridinyl-methyl)-N-methylamino]benzoyl]-Glu-Gly-bAla}₄-Lys₂-Lys-bAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 28)

The above product was treated for 1.5 h at room temperature withphenol/H₂O/thioanisole/1,2-dithioethane/CF₃COOH, 0.75:0.5:0.5:0.25:10,(12 mL). Resin residue was then filtered off and washed on a sinter withsmall aliquots of neat CF₃COOH. The combined filtrate and washings weretreated with Et₂O (100 mL) and cooled. The precipitated product wascollected by centrifugation and the ethereal supernatant was decanted.The product was washed three more times with Et₂O in a similar fashion.The final crude product was dried in vacuo (61 mg orange powder). Thismaterial was redissolved in 4 mL 0.1% aq CF₃COOH and filtered. Theresulting solution was applied (two separate runs) to an RP-HPLC column(Vydac 218TP1022; 22×250 mm). The column was eluted at 9 mL/min using agradient from 17.5 to 27.5% MeCN in 0.1% aq CF₃COOH over 40 min (25°C.). Peak fractions were collected, monitored (analytical RP-HPLC) andpooled as appropriate. After vacuum centrifugation, pure title compound(13.5 mg) was obtained. Anal. RP-HPLC: t_(R)=17.8 min (Vydac 218TP54,17.5-27.5% MeCN in 0.1% aq CF₃COOH over 20 min, 1 mL/min, 25° C.;purity>99%, l=200-300 nm). DE MALDI-TOF MS: [M+H]⁺=4962(C₂₂₅H₃₂₁N₈₁O₄₈S=4960.54).

Example 2aH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27)

H-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-Resin(SEQ ID No. 1) (see example 1; 411 mg, 0.075 mmol) was acylated withFmoc-Cys(Trt)-OH (264 mg, 0.45 mmol), PyBOP (234 mg, 0.45 mmol), HOBt(61 mg, 0.45 mmol) and P^(i) ₂NEt (0.12 mL, 0.675 mmol) in DMF (3 mL)during 3 h. The resulting peptidyl resin was washed with DMF (3×5 min,25 mL each), drained and treated with 20% piperidine in DMF during 20min. After filtration of the reagent, the productH-Cys(Trt)-Arg(Pmc)-Gln(Trt)-Ile-Lys(Boc)-Ile-Trp-Phe-Gln(Trt)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-Met-Lys(Boc)-Trp-Lys(Boc)-Lys(Boc)-Resin(SEQ ID No. 27) was washed successively with DMF, CH₂Cl₂ and Et₂O,before being dried in vacuo.

The above product was treated for 2 h at room temperature withphenol/H₂O/thioanisole/1,2-dithioethane/CF₃COOH, 0.75:0.5:0.5:0.25:10(12 mL). Resin residue was then filtered off and washed on a sinter withsmall aliquots of neat CF₃COOH. The combined filtrate and washings weretreated with Et₂O (100 mL) and cooled. The precipitated product wascollected by centrifugation and the ethereal supernatant was decanted.The product was washed three more times with Et₂O in a similar fashion.The final crude product was dried in vacuo (238 mg). An aliquot (119 mg)of this material was redissolved in 2 mL 0.1% aq CF₃COOH and filtered.The resulting solution was applied to an RP-HPLC column (Vydac218TP1022; 22×250 mm). The column was eluted at 9 mL/min using agradient from 17.5 to 27.5% MeCN in 0.1% aq CF₃COOH over 40 min (25°C.). Peak fractions were collected, monitored (analytical RP-HPLC) andpooled as appropriate. After vacuum centrifugation, pure title compound(60.9 mg) was obtained. Anal. RP-HPLC: t_(R)=15.8 min (Vydac 218TP54,17.5-27.5% MeCN in 0.1% aq CF₃COOH over 20 min, 1 mL/min, 25° C.;purity>99%, l=214 nm). DE MALDI-TOF MS: [M+H]⁺=2351(C₁₀₇H₁₇₃N₃₅O₂₁S₂=2349.87).

N-[3-(Maleimido)benzoyl]-doxorubicin

Doxorubicin hydrochloride (Aldrich, 86,036-0; 5.9 mg, 0.01 mmol) wasdissolved in H₂O (1 mL) and DMF (0.5 mL). Buffer (0.1 M aq phosphate, pH7.2; 0.5 mL) was added with stirring. To the resulting suspension3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma, M2786; 12.9mg, 0.04 mmol) in DMF (1 mL) was added dropwise. The red-colouredreaction mixture cleared temporarily and after ca. 10 min precipitationwas observed. Reaction progress was monitored by anal. RP-HPLC and after2 h all doxorubicin had reacted. The mixture was then diluted with H₂O(1.5 mL), cooled to 4° C. and centrifuged. The supernatant was decanted.The residual pellet was redissolved in DMF (1 mL) and diluted with 0.1%aq CF₃COOH (2 mL). This solution was applied to a solid-phase extractioncartridge (LiChrolut RP-18, 500 mg; Merck); the cartridge was washedwith 0.1% aq CF₃COOH (4 mL) and eluted with 6:4 MeCN/H₂O (containing0.1% CF₃COOH) in two fractions (2×4 mL). The first fraction containedthe title compound and was used directly in the next step.

N-{3-[3-(Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)succinimido]benzoyl}-doxorubicin(SEQ ID No. 27)

The above N-[3-(Maleimido)benzoyl]-doxorubicin solution was diluted withDMF (1 mL) and Et₃N (50 mL) was added. The solution turned dark brown.H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (5 mg), dissolved in DMF (1 mL) was then added. Themixture was stirred and the brown colour was observed to discharge,leaving a light red solution. The reaction was monitored by anal.RP-HPLC. After 1.5 h, all 3-(maleimido-benzoyl)-doxorubicin had reacted.The solution was acidified with AcOH (0.5 mL), diluted with H₂O (3 mL)and applied to a solid-phase extraction cartridge (LiChrolut RP-18, 500mg; Merck). The cartridge was washed with 0.1% aq CF₃COOH (6 mL) andeluted (6 mL of 6:4 MeCN/H₂O (containing 0.1% CF₃COOH)). The eluate wasdried by vacuum centrifugation. The residue was redissolved in 0.1% aqCF₃COOH (2 mL), filtered and applied to an RP-HPLC column (Vydac218TP1022; 22×250 mm). The column was eluted at 9 mL/min using agradient from 20 to 40% MeCN in 0.1% aq CF₃COOH over 40 min (25° C.).Peak fractions were collected, monitored (analytical RP-HPLC) and pooledas appropriate. After vacuum centrifugation, pure title compound (1.2mg) was obtained. Anal. RP-HPLC: t_(R)=15.6 & 15.8 (partly resolvedthioether diastereomers) min (Vydac 218TP54, 0-60% MeCN in 0.1% aqCF₃COOH over 20 min, 1 mL/min, 25° C.; purity>95%, l=200-300 nm). DEMALDI-TOF MS: [M+H]⁺=3094, [M+2H]²⁺=1548 (C₁₄₅H₂₀₇N₃₇O₃₅S₂=3092.56).

Example 3a 2′-[(3-Maleimidopropionoyl)]-paclitaxel

3-Maleimidopropionic acid (5.7 mg, 0.034 mmol) was dissolved in dryCH₂Cl₂ (0.5 mL). The mixture was stirred and diisopropylcarbodiimide(2.4 mg, 0.019 mmol) in dry CH₂Cl₂ (0.5 mL) was added. The reaction wasallowed to proceed with stirring for 30 min. Solvent was then removedunder reduced pressure. The residue of 3-maleimidopropionic acidanhydride was redissolved in dry pyridine (0.5 mL). A solution ofpaclitaxel (Aldrich 41,701-7; 1 mg, 0.0012 mmol) in dry pyridine (0.5mL) was added and the mixture was stirred under N₂ for 3 h. It was thenevaporated to dryness under reduced pressure. The residue was treatedwith H₂O (1.5 mL). After 10 min, it was extracted with CH₂Cl₂ (3×5 mL).The combined extracts were washed with H₂O (3×1 mL), dried with MgSO₄,filtered and evaporated to dryness to leave a white residue of the titlecompound.

2′-{3-[3-(Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)succinimido]propionoyl}-paclitaxel(SEQ ID No. 27)

The product from the previous reaction was redissolved in DMF (0.25 mL)andH-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (2.5 mg, 0.0011 mmol), dissolved in DMF (0.25 mL) wasthen added, together with Et₃N (ca. 0.05 mL). The mixture was stirredunder N₂ and was monitored by anal. RP-HPLC. After 45 min, the reactionwas complete. The mixture was diluted to 2 mL with 0.1% aq CF₃COOH,filtered and applied to an RP-HPLC column (Vydac 218TP1022; 22×250 mm).The column was eluted at 9 mL/min using a gradient from 0 to 60% MeCN in0.1% aq CF₃COOH over 40 min (25° C.). Peak fractions were collected,monitored (analytical RP-HPLC) and pooled as appropriate. After vacuumcentrifugation, pure title compound (1.2 mg) was obtained. Anal.RP-HPLC: t_(R)=17.4 & 17.5 (partly resolved thioether diastereomers) min(Vydac 218TP54, 0-60% MeCN in 0.1% aq CF₃COOH over 20 min, 1 mL/min, 25°C.; purity>95%, l=200-300 nm). DE MALDI-TOF MS: [M+H]⁺=3356,[M+2H]²⁺=1679 (C₁₆₁H₂₂₉N₃₇O₃₈S₂=3354.90).

Example 4a In vitro cytotoxic activity of{[4[N-(2,4-diamino-6-pteridinyl-methyl)-N-methylamino]benzoyl]-Glu-Gly-bAla}₄-Lys₂-Lys-bAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 28)

This compound (abbreviated ‘MTX-Pen’ in tables below) was evaluated forits ability to inhibit cell proliferation of normal (immortalised) humancells (HaCaT cells, Tables 1 & 2) and a human colorectal cancer cellline (HT29, Table 3). The free drug methotrexate (Tables 1-3) and thefree vectorH-Ala-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 28) (abbreviated ‘Pen’ in Table 1 below) were included forthe purposes of comparison.

Assay procedure—Cells were seeded into 96-well plates at 2,500cells/well in DMEM with 10% foetal calf serum and antibiotics. Afterovernight incubation, test compound dilutions in cell medium wereprepared and were added to the cells. Samples were taken 1, 2, 3, and 4days after compound addition. Nucleotide Releasing Reagent (LumiTechViaLight kit) was added in order to lyse the cells and release ATP.After incubation at room temperature (5 min), the mixtures weretransferred to opaque 96-well plates and stored at −20° C. untilanalysis. After loading plates into a luminometer (Lucy 1, LabtechInternational), ATP Monitoring Reagent (20 mL/well, LumiTech ViaLightkit) was added to each well successively and light intensity wasmeasured immediately. Six readings were taken per sample. Each assaypoint was established using six replicates and appropriate controls. ATPbioluminescence was found to be proportional to viable cell count overthe entire cells/well range used.

Statistically significant results in the tables below are printed inbold face.

TABLE 1 (HaCaT Cells) % Cell Death Day 1 Day 2 Day 3 Day 4 Dose (μM) MTXMTX-Pen Pen MTX MTX-Pen Pen MTX MTX-Pen Pen MTX MTX-Pen Pen 40.0 4 29 1615 82 −22 79 97 5 92 98 12 13.3 22 −42 18 35 63 0 82 97 −17 92 98 −6 4.44 −8 8 24 45 −4 77 95 −1 93 98 10 1.5 13 −24 16 31 82 −31 77 82 2 94 88−14 0.5 −4 −19 6 31 2 −6 75 29 −29 93 49 −26 0.2 7 14 26 11 21 0 79 20−3 93 51 21

TABLE 2 (HaCaT Cells) % Cell Death Day 1 Day 2 Day 3 Day 4 Dose MTX-MTX- MTX- MTX- (μM) MTX Pen MTX Pen MTX Pen MTX Pen 40.0 42 88 95 9413.3 27 87 95 94 4.4 21 15 70 52 97 95 92 88 1.5 14 19 67 12 96 −16 9117 0.5 0 13 59 24 96 −27 91 2 0.2 3 41 94 86 0.1 19 7 45 65

TABLE 3 (HT 29 Cells) % Cell Death Day 1 Day 2 Day 3 Day 4 Dose MTX-MTX- MTX- MTX- (μM) MTX Pen MTX Pen MTX Pen MTX Pen 40.0 31 79 96 9813.3 3 45 88 96 4.4 −14 10 −4 6 58 46 86 77 1.5 17 16 −5 9 48 15 84 450.5 15 14 −12 8 52 17 88 16 0.2 10 −5 54 85 0.1 6 −17 52 84

Example 5a [(3-Maleimidopropionoyl)]bohemine

3-Maleimidopropionic acid (12.8 mg, 76 mmol) was dissolved in CH₂Cl₂ (1mL). The mixture was stirred and DIC (5.3 mg, 42 mmol) in dry CH₂Cl₂(0.5 mL) was added. The reaction was allowed to proceed with stirringfor 40 min. Solvent was then removed under reduced pressure. The residueof 3-maleimidopropionic acid anhydride was redissolved in dry pyridine(0.5 mL). A solution of bohemine({6-(benzylamino)-2-[(3-(hydroxypropyl)amino]-9-isopropylpurine}, 10.3mg, 30 mmol) and DMAP (0.35 mg, 2 mmol) in dry pyridine (0.5 mL) wasadded and the mixture was stirred under N₂ for 1 h. It was thenevaporated to dryness under reduced pressure. The residue wasredissolved in DMF (1 mL) and applied to an RP-HPLC column (Vydac218TP1022; 22×250 mm). The column was eluted at 9 mL/min using agradient from 10-60% MeCN gradient in 0.1% aq. CF₃COOH over 40 min (25°C.). Peak fractions were collected, monitored (Anal. RP-HPLC) and pooledas appropriate. After vacuum centrifugation, pure title compound (14.7mg, 87.8%) was obtained. Anal. RP-HPLC: t_(R)=17.7 min (column (Vydac218TP54, 0-60% MeCN in 0.1% aq. CF₃COOH over 20 min, 1 mL/min., 25° C.;purity>95%, λ=200-300 nm). ¹H-NMR (CDCl₃) and DE MALDI-TOF MS spectrawere consistent with the proposed structure (C₂₅H₂₉N₇O₄=491.54).

O-{3-[3-(Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)succinimido]propionoyl}-bohemine(SEQ ID No. 27)

The product from the previous reaction (0.74 mg, 1.5 mmol) was dissolvedin DMF (0.3 mL) and Et₃N (50 mL) was added.H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH(SEQ ID No. 27) (3.5 mg, 1.5 mmol), dissolved in DMF (0.25 mL) was thenadded. The mixture was stirred under N₂ and was monitored by anal.RP-HPLC. After 1 h, the reaction was complete. The mixture was filteredand applied to an RP-HPLC column (Vydac 218TP1022; 22×250 mm). Thecolumn was eluted at 9 mL/min using a gradient from 10-60% MeCN gradientin 0.1% aq. CF₃COOH over 40 min (25° C.). Peak fractions were collected,monitored (Anal. RP-HPLC) and pooled as appropriate. After vacuumcentrifugation, pure title compound (1.7 mg, 40%) was obtained. Anal.RP-HPLC: t_(R)=15.0 min (Vydac 218TP54, 0-60% MeCN in 0.1% aq. CF₃COOHover 20 min, 1 mL/min., 25° C.; purity>95%, λ=200-300 nm). DE MALDI-TOFMS: [M+H]⁺=2842 (C₁₃₂H₂₀₂N₄₂O₂₅S₂=2841.42).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A macromolecule comprising a drug moiety linked to a carrier moiety,wherein (a) the drug moiety is not a peptide, an oligonucleotide,cholesterol or biotin and is selected from the group consisting of acardioprotective drug, an anti-arrhythmic drug, an anti-inflammatorydrug, a neuroleptic drug, an anti-convulsant drug, an anxio-lytic drug,an antiviral drug, a diagnostic and a tri-substituted purine; (b) thecarrier moiety is a homeobox peptide derived from the helix 3 sequenceof the pAntp peptide or fragment thereof; and wherein the carrier moietyfacilitates the cellular internalization of the drug moiety.
 2. Themacromolecule of claim 1, wherein the drug moiety is a cardioprotectivedrug.
 3. The macromolecule of claim 1, wherein the drug moiety is ananti-arrhythmic drug.
 4. The macromolecule of claim 1, wherein the drugmoiety is an anti-inflammatory drug.
 5. The macromolecule of claim 1,wherein the drug moiety is a neuroleptic drug.
 6. The macromolecule ofclaim 1, wherein the drug moiety is an anti-convulsant drug.
 7. Themacromolecule of claim 1, wherein the drug moiety is an anxio-lyticdrug.
 8. The macromolecule of claim 1, wherein the drug moiety is anantiviral drug.
 9. The macromolecule of claim 1, wherein the drug moietyis a diagnostic.
 10. The macromolecule of claim 1, wherein the drugmoiety is a tri-substituted purine selected from the group consisting ofolomoucine, roscovitine and bohemine.
 11. The macromolecule of claim 1,wherein the carrier moiety comprises a peptide having the amino acidsequence set forth in SEQ ID NO
 2. 12. The macromolecule of claim 1,wherein the carrier moiety further comprises a cysteine as a terminalresidue.
 13. The macromolecule of claim 1, wherein the carrier moietycomprises a carboxy terminal amino acid residue in which the carboxylgroup is converted into a carboxamide group.
 14. The macromolecule ofclaim 1, wherein the carrier moiety is comprised of D-amino acids. 15.The macromolecule of claim 1, wherein the carrier moiety is inretro-inverso form.
 16. A macromolecule comprising a drug moiety linkedto a carrier moiety, wherein (a) the drug moiety is not a peptide, anoligonucleotide, cholesterol or biotin; (b) the carrier moiety is ahomeobox peptide derived from the helix 3 sequence of the pAntp peptideor fragment thereof; and (c) the drug moiety is linked to the carriermoiety by way of a linker moiety; wherein the carrier moiety facilitatesthe cellular internalization of the drug moiety.
 17. The macromoleculeof claim 16, wherein the linker moiety is selected from the groupconsisting of bi-functional alkyl, aryl, aralkyl and peptidic moieties;multi-functional alkyl, aryl, aralkyl and peptidic moieties; alkyl, aryland aralkyl aldehydes; alkyl, aryl and aralkyl acids; alkyl, aryl andaralkyl esters; alkyl, aryl and aralkyl anhydrides; sulphydryl groups,and carboxyl groups.
 18. The macromolecule of claim 16, wherein thelinker moiety is selected from the group consisting of maleimido benzoicacid derivatives, maleimido propionic acid derivatives and succinimidoderivatives.
 19. The macromolecule of claim 16 wherein the linker moietyis derived from the group consisting of cyanuric bromide, cyanuricchloride, carbonyldiimidazole, succinimidyl esters and sulphonichalides.
 20. The macromolecule of claim 16, wherein the linker moiety isselected from the group consisting of (methylamino)benzoyl-Cys,succinimidobenzoyl-Cys, succinimidopropionoyl-Cys, β-alanyl-succinyl,acetyl-Cys and (4″-aminoanilino)-succinimidopropionoyl-Cys.
 21. Themacromolecule of claim 16, wherein the linker moiety comprises one tofour amino acids selected from the group consisting of cysteine,glycine, glutamic acid and β-alanine.
 22. The macromolecule of claim 16,wherein the linker moiety comprises a cysteine residue.
 23. Themacromolecule of claim 22, wherein the linker moiety further comprisesβ-alanine.
 24. The macromolecule of claim 22, wherein the linker moietyfurther comprises glycine.
 25. The macromolecule of claim 16, whereinthe linker moiety is a network of lysine residues.
 26. The macromoleculeof claim 16, wherein the carrier moiety bears more than one drug moiety.27. The macromolecule of claim 26, wherein the drug moieties aredifferent.
 28. The macromolecule of claim 26, wherein each drug moietyis linked to the carrier moiety by way of an identical linker moiety.29. The macromolecule of claim 26, wherein each drug moiety is linked tothe carrier moiety by a different linker moiety.
 30. A macromoleculecomprising a drug moiety covalently bound to a carrier moiety, wherein(a) the drug moiety is not a peptide, an oligonucleotide, cholesterol orbiotin and is selected from the group consisting of a cardioprotectivedrug, an anti-arrhythmic drug, an anti-inflammatory drug, a neurolepticdrug, an anti-convulsant drug, an anxio-lytic drug, an antiviral drug, adiagnostic and a tri-substituted purine; and (b) the carrier moiety is aderivative of a homeobox peptide or fragment thereof comprising theamino acid sequence RRMKWKK (SEQ ID No:2), wherein (i) one or two aminoacid residues are replaced by a naturally or non-naturally occurringamino acid residue; (ii) there is an insertion of one or moremultivalent amino acids; (iii) one or more lysine residues are attachedto at least one end; (iv) there is a cysteine as a terminal residue; or(v) any combination of (i)-(iv); wherein the carrier moiety facilitatesthe cellular internalization of the drug moiety.
 31. A macromoleculecomprising a drug moiety linked to a carrier moiety, wherein (a) thedrug moiety is not a peptide, an oligonucleotide, cholesterol or biotin;(b) the carrier moiety is a derivative of a homeobox peptide or fragmentthereof comprising the amino acid sequence RRMKWKK (SEQ ID No:2),wherein (i) one or two amino acid residues are replaced by a naturallyor non-naturally occurring amino acid residue; (ii) there is aninsertion of one or more multivalent amino acids; (iii) one or morelysine residues are attached to at least one end; (iv) there is acysteine as a terminal residue; or (v) any combination of (i)-(iv); and(c) the drug moiety is linked to the carrier moiety by way of a linkermoiety; wherein the carrier moiety facilitates the cellularinternalization of the drug moiety.
 32. The macromolecule of claim 10,wherein the tri-substituted purine is roscovitine.
 33. The macromoleculeof claim 30, wherein one or two amino acid residues are replaced by anaturally or non-naturally occurring amino acid residue.
 34. Themacromolecule of claim 30, wherein one or more lysine residues areattached to at least one end.
 35. The macromolecule of claim 30, whereinone or more lysine residues are attached to at least one end.
 36. Themacromolecule of claim 30, wherein there is a cysteine as a terminalresidue.
 37. The macromolecule of claim 31, wherein one or two aminoacid residues are replaced by a naturally or non-naturally occurringamino acid residue.
 38. The macromolecule of claim 31, wherein there isan insertion of one or more multivalent amino acids.
 39. Themacromolecule of claim 31, wherein one or more lysine residues areattached to at least one end.
 40. The macromolecule of claim 31, whereinthere is a cysteine as a terminal residue.